Deep immune profiling of chronic rhinosinusitis in allergic and non-allergic cohorts using mass cytometry.

Chronic rhinosinusitis (CRS) is a persistent nasal and paranasal sinus mucosa inflammation comprising two phenotypes, namely CRS with nasal polyps (CRSwNP) and without (CRSsNP). CRSwNP can be associated with asthma and hypersensitivity to non-steroidal anti-inflammatory drug (NSAID) in a syndrome known as NSAID-exacerbated respiratory disease (N-ERD). Furthermore, CRS frequently intertwines with respiratory allergies. This study investigated levels of 33 different nasal and serum cytokines and phenotypic characteristics of peripheral blood mononuclear cells (PBMCs) within cohorts of CRS patients (n = 24), additionally examining the influence of comorbid respiratory allergies by mass cytometry. N-ERD patients showed heightened type 2 nasal cytokine levels. Mass cytometry revealed increased activated naive B cell levels in CRSwNP and N-ERD, while resting naive B cells were higher in CRSsNP. Th2a cell levels were significantly elevated in allergic subjects, but not in CRS groups. In conclusion, there are distinct immunological features in PBMCs of CRS phenotypes and allergy.

Unravelling the Immunity of Poultry Against the Extracellular Protozoan Parasite Histomonas meleagridis Is a Cornerstone for Vaccine Development: A Review.

The protozoan parasite Histomonas meleagridis is the causative agent of histomonosis in gallinaceous birds, predominantly in turkeys and chickens. Depending on the host species the outcome of the disease can be very severe with high mortality as observed in turkeys, whereas in chickens the mortality rates are generally lower. The disease is known for more than 100 years when in vitro and in vivo investigations started to understand histomonosis and the causative pathogen. For decades histomonosis could be well-controlled by effective drugs for prevention and therapy until the withdrawal of such chemicals for reasons of consumer protection in Europe, the USA and additional countries worldwide. Consequently, research efforts also focused to find new strategies against the disease, resulting in the development of an efficacious live-attenuated vaccine. In addition to efficacy and safety several studies were performed to obtain a deeper understanding of the immune response of the host against H. meleagridis. It could be demonstrated that antibodies accumulate in different parts of the intestine of chickens following infection with H. meleagridis which was much pronounced in the ceca. Furthermore, expression profiles of various cytokines revealed that chickens mounted an effective cecal innate immune response during histomonosis compared to turkeys. Studying the cellular immune response following infection and/or vaccination of host birds showed a limitation of pronounced changes of B cells and T-cell subsets in vaccinated birds in comparison to non-protected birds. Additionally, numbers of lymphocytes including cytotoxic T cells increased in the ceca of diseased turkeys compared to infected chickens suggesting an immunopathological impact on disease pathogenesis. The identification of type 1 and type 2 T-helper (Th) cells in infected and lymphoid organs by in situ hybridization did not show a clear separation of Th cells during infection but revealed a coherence of an increase of interferon (IFN)-γ mRNA positive cells in ceca and protection. The present review not only summarizes the research performed on the immune response of host birds in the course of histomonosis but also highlights the specific features of H. meleagridis as a model organism to study immunological principles of an extracellular organism in birds.

Allocation of Interferon Gamma mRNA Positive Cells in Caecum Hallmarks a Protective Trait Against Histomonosis.

Histomonosis is a parasitic disease of gallinaceous birds characterized by necrotic lesions in cacum and liver that usually turns fatal in turkeys while it is less severe in chickens. Vaccination using in vitro attenuated Histomonas meleagridis has been experimentally shown to confer protection against histomonosis. The protective mechanisms that underpin the vaccine-induced immune response are not resolved so far. Therefore, the actual study aimed to evaluate the location and quantitative distribution patterns of signature cytokines of type 1 [interferon gamma (IFN-γ)] or type 2 [interleukin (IL)-13] immune responses in vaccinated or infected hosts. An intergroup and interspecies difference in the spatial and temporal distribution patterns of cytokine mRNA positive cells was evident. Quantification of cells showed a significantly decreased percentage of IFN-γ mRNA positive cells at 4 days post-inoculation (DPI) in caeca of turkeys inoculated exclusively with the attenuated or the virulent inocula, compared to control birds. The decrement was followed by a surge of cells expressing mRNA for IFN-γ or IL-13, reaching a peak of increment at 10 DPI. By contrast, turkeys challenged following vaccination showed a slight increment of cecal IFN-γ mRNA positive cells at 4 DPI after which positive cell counts became comparable to control birds. The increase in infected birds was accompanied by an extensive distribution of positively stained cells up to the muscularis layer of cecal tissue whereas the vaccine group maintained an intact mucosal structure. In chickens, the level of changes of positive cells was generally lower compared to turkeys. However, control chickens were found with a higher percentage of IFN-γ mRNA positive cells in cecum compared to their turkey counterparts indicating a higher resistance to histomonosis, similar to the observation in immunized turkeys. In chickens, it could be shown that the changes of cytokine-positive cells were related to variations of mononuclear cells quantified by immunofluorescence. Furthermore, gene expression measured by reverse transcription quantitative real time PCR confirmed variations in organs between the different groups of both bird species. Overall, it can be concluded that a proportionally increased, yet controlled, allocation of IFN-γ mRNA positive cells in caeca hallmarks a protective trait against histomonosis.

Vaccination against histomonosis limits pronounced changes of B cells and T-cell subsets in turkeys and chickens.

The protozoan parasite Histomonas meleagridis is the causative agent of histomonosis in gallinaceous birds. In turkeys, the disease can result in high mortality due to severe inflammation and necrosis in caecum and liver, whereas in chickens the disease is less severe. Recently, experimental vaccination was shown to protect chickens and turkeys against histomonosis but dynamics in the cellular immune response are not yet demonstrated. In the present work, different groups of birds of both species were vaccinated with attenuated, and/or infected with virulent histomonads. Flow cytometry was applied at different days post inoculation to analyse the absolute number of T-cell subsets and B cells in caecum, liver, spleen and blood, in order to monitor changes in these major lymphocyte subsets. In addition, in chicken samples total white blood cells were investigated. Infected turkeys showed a significant decrease of T cells in the caecum within one week post infection compared to control birds, whereas vaccination showed delayed changes. The challenge of vaccinated turkeys led to a significant increase of all investigated lymphocytes in the blood already at 4 DPI, indicating an effective and fast recall response of the primed immune system. In the caecum of chickens, changes of B cells, CD4+ and CD8α+ T cells were much less pronounced than in turkeys, however, mostly caused by virulent histomonads. Analyses of whole blood in non-vaccinated but infected chickens revealed increasing numbers of monocytes/macrophages on all sampling days, whereas a decrease of heterophils was observed directly after challenge, suggesting recruitment of this cell population to the local site of infection. Our results showed that virulent histomonads caused more severe changes in the distribution of lymphocyte subsets in turkeys compared to chickens. Moreover, vaccination with attenuated histomonads resulted in less pronounced alterations in both species, even after challenge.

In situ hybridization to detect and localize signature cytokines of T-helper (Th) 1 and Th2 immune responses in chicken tissues.

The avian immune system has been shown to possess a repertoire of cytokines directing T-helper (Th) 1 and Th2 types of immune responses similar to that in mammals. The objective of this study was to establish in situ hybridization (ISH) for the localization of mRNA of selected signal cytokines, chicken interferon-γ (ChIFN-γ), chicken interleukin (ChIL)-4 and ChIL-13 in fixed tissues. RNA probes were generated to hybridize to 488, 318, and 417bp of the respective target mRNA. Probe concentrations ranging from 100ng/ml to 400ng/ml were shown to be suitable to label cells that expressed these cytokines. The specificity of every probe was verified using the respective sense probe. ChIFN-γ, ChIL-4 and ChIL-13 positive cells were observed in the lymphocytic infiltrations of liver and in the periarteriolar lymphatic sheaths of spleen collected from specific-pathogen-free chickens. ISH of these cytokines in a severely inflamed liver due to infiltration with the parasite Histomonas meleagridis revealed the expression of both ChIFN-γ and ChIL-13 mRNA in the mononuclear infiltrates. In conclusion, ChIFN-γ, ChIL-4 and ChIL-13 mRNA were efficiently localized by ISH, which supplies a valid technique to characterize immune responses in fixed tissues.