The aim of this study was to investigate the effect of regulatory T cells (Tregs) on B-cell immune responses against outer membrane protein (OMP) from nontypeable Haemophilus influenzae (NTHi) in vitro, to clarify its exact mechanism from an immunologic standpoint. Mice were vaccinated intranasally with OMP to induce OMP-specific immune responses in the nasal mucosa. Mononuclear cells (MNCs) were collected from the nasal mucosa, and Tregs and helper T (Th) cells were isolated separately from the spleens of those mice. Three different cell culture groups were allocated: MNCs cocultured with Tregs, MNCs cocultured with Th cells, and MNCs cultured alone. At 24 and 72 hours after cell culture, the concentrations of various cytokines and antibodies in culture supernatants were measured to assess the effects of Tregs and Th cells on B-cell responses. Cytokine levels and specific anti-OMP antibody levels in culture media were determined using enzyme-linked immunosorbent assay. CD69 or CD80 expression on B220-positive cells was detected using flow cytometric analysis. Th1 and Th2 cytokine concentrations were significantly elevated in the 3 groups incubated with OMP from 24 to 72 hours. Additionally, interleukin-10 levels were significantly higher in the Treg and Th groups than in the control group. Levels of OMP-specific immunoglobulin A did not differ significantly among the groups. The ratios of CD69+B220+ B2 cells were nearly the same in the 3 groups; however, the ratio of CD80+B220+ B2 cells was higher in the control group than in the Treg and Th groups during incubation. Tregs and Th cells did not affect OMP-specific immunoglobulin A production in this study. However, these cells may partially inhibit B-cell functions, such as T-cell activation. These inhibitory effects may be related to interleukin-10.
Contribution of LTi and TH17 cells to B cell aggregate formation in the central nervous system in a mouse model of multiple sclerosis
In a subgroup of patients suffering from progressive multiple sclerosis (MS), which is an inflammation-mediated neurodegenerative disease of the central nervous system (CNS), B cell aggregates were discovered within the meninges. Occurrence of these structures was associated with a more severe disease course and cortical histopathology. We have developed the B cell-dependent MP4-induced experimental autoimmune encephalomyelitis (EAE) as a mouse model to mimic this trait of the human disease. The aim of this study was to determine a potential role of lymphoid tissue inducer (LTi) and TH17 cells in the process of B cell aggregate formation in the MP4 model. We performed flow cytometry of cerebellar and splenic tissue of MP4-immunized mice in the acute and chronic stage of the disease to analyze the presence of CD3-CD5-CD4+RORγt+ LTi and CD3+CD5+CD4+RORγt+ TH17 cells. Myelin oligodendrocyte glycoprotein (MOG):35-55-induced EAE was used as B cell-independent control model. We further determined the gene expression profile of B cell aggregates using laser capture microdissection, followed by RNA sequencing. While we were able to detect LTi cells in the embryonic spleen and adult intestine, which served as positive controls, there was no evidence for the existence of such a population in acute or chronic EAE in neither of the two models. Yet, we detected CD3-CD5-CD4-RORγt+ innate lymphoid cells (ILCs) and TH17 cells in the CNS, the latter especially in the chronic stage of MP4-induced EAE. Moreover, we observed a unique gene signature in CNS B cell aggregates compared to draining lymph nodes of MP4-immunized mice and to cerebellum as well as draining lymph nodes of mice with MOG:35-55-induced EAE. The absence of LTi cells in the cerebellum suggests that other cells might take over the function as an initiator of lymphoid tissue formation in the CNS. Overall, the development of ectopic lymphoid organs is a complex process based on an interplay between several molecules and signals. Here, we propose some potential candidates, which might be involved in the formation of B cell aggregates in the CNS of MP4-immunized mice.
USP16-mediated deubiquitination of calcineurin A controls peripheral T cell maintenance
... and sex-matched mice were immunized s.c. with MOG35-55 peptide (300 μg) mixed in CFA (Sigma-Aldrich) containing 5 mg/ml heat-killed Mycobacterium tuberculosis H37Ra (Difco). Pertussis toxin (200 ng, List Biological Laboratories) in PBS was administered i.v. on days 0 and 2. Mice were examined daily and scored for disease severity using the standard scale: 0, no clinical signs; 1, limp tail; 2, pa ... and sex-matched mice were immunized s.c. with MOG35-55 peptide (300 μg) mixed in CFA (Sigma-Aldrich) containing 5 mg/ml heat-killed Mycobacterium tuberculosis H37Ra (Difco). Pertussis toxin (200 ng, List Biological Laboratories) in PBS was administered i.v. on days 0 and 2. Mice were examined daily and scored for disease severity using the standard scale: 0, no clinical signs; 1, limp tail; 2, pa
Calcineurin acts as a calcium-activated phosphatase that dephosphorylates various substrates, including members of the nuclear factor of activated T cells (NFAT) family, to trigger their nuclear translocation and transcriptional activity. However, the detailed mechanism regulating the recruitment of NFATs to calcineurin remains poorly understood. Here, we report that calcineurin A (CNA), encoded by PPP3CB or PPP3CC, is constitutively ubiquitinated on lysine 327, and this polyubiquitin chain is rapidly removed by ubiquitin carboxyl-terminal hydrolase 16 (USP16) in response to intracellular calcium stimulation. The K29-linked ubiquitination of CNA impairs NFAT recruitment and transcription of NFAT-targeted genes. USP16 deficiency prevents calcium-triggered deubiquitination of CNA in a manner consistent with defective maintenance and proliferation of peripheral T cells. T cell-specific USP16 knockout mice exhibit reduced severity of experimental autoimmune encephalitis and inflammatory bowel disease. Our data reveal the physiological function of CNA ubiquitination and its deubiquitinase USP16 in peripheral T cells. Notably, our results highlight a critical mechanism for the regulation of calcineurin activity and a novel immunosuppressive drug target for the treatment of autoimmune diseases.
Noradrenergic terminal density varies among different groups of hypoglossal premotor neurons
Boyle, CE;Parkar, A;Barror, A;Kubin, L;
Journal Of Chemical Neuroanatomy100101651May 22, 2019
Product: Cholera Toxin B Subunit (Choleragenoid)
from Vibrio cholerae in Low Salt
; - 2.2. Retrograde tracer injections. Injections of cholera toxin beta sub-unit (CTb; List Biological Laboratories, Campbell, CA) were used to retrogradely label premotor cells with axonal projections to the XII motor nucleus, as described previously [Rukhadze & Kubin, 2007a
- One hour after the first immunisation, pertussis toxin (500 ng in 100 μl saline; List Biological Laboratories, Campbell, CA, USA) was intraperitoneally injected into each animal. 2.3. Cell preparation and flow cytometry. Triceps