Lymphangiogenesis associated with tertiary lymphoid structure (TLS) has been reported in numerous studies. However, the kinetics and dynamic changes occurring to the lymphatic vascular network during TLS development have not been studied. Using a viral-induced, resolving model of TLS formation in the salivary glands of adult mice we demonstrate that the expansion of the lymphatic vascular network is tightly regulated. Lymphatic vessel expansion occurs in two distinct phases. The first wave of expansion is dependent on IL-7. The second phase, responsible for leukocyte exit from the glands, is regulated by lymphotoxin (LT)βR signaling. These findings, while highlighting the tight regulation of the lymphatic response to inflammation, suggest that targeting the LTα1β2/LTβR pathway in TLS-associated pathologies might impair a natural proresolving mechanism for lymphocyte exit from the tissues and account for the failure of therapeutic strategies that target these molecules in diseases such as rheumatoid arthritis.
Background Tertiary lymphoid organ formation is a hallmark of many chronic inflammatory diseases such as Sjögren’s syndrome and rheumatoid arthritis. The processes underlying the persistence of inflammation in these ectopic lymphoid aggregates remain to be elucidated and, if better understood, could shed light on therapeutic targets aimed at diverting the immune response away from chronicity and towards resolution. It has previously been proposed that an excess of antigen may result in the detrimental prolongation of the local immune response and persistence of ectopic lymphoid organs within the tissue. Objectives Here, we aim to test this hypothesis by expanding an existing novel murine model using adenovirus-infected salivary glands to address the factors influencing memory formation and contributing to the persistence of tertiary lymphoneogenesis (1). Methods The submandibular glands of female C57BL/6 mice were cannulated via the excretory duct with 108 p.f.u. of luciferase-expressing replication-deficient adenovirus. At day 15 post-cannulation, the mice were recannulated and then culled at days 2, 5, 8 and 15 post-recannulation. Flow cytometry was carried out on collagenase-digested salivary glands to determine the dynamics of the lymphoid-like stromal cell (LLSc) (CD45-EPCAM-CD31-gp38+) response. Real-time PCR and immunofluorescence were used to quantify mRNA expression of lymphoid chemokines and visually characterise aggregate formation respectively. ELISAs were used to quantify the anti-viral response (IgG and IgM) in the serum. Results As a result of increased initial proliferation, LLSc activation remained high at day 8 post-recannulation instead of beginning to resolve as seen in historical single cannulated control mice. Expression of lymphoid chemokines CXCL13 and CCL19 were significantly higher at days 2, 5 and 8, accompanied by higher expression of AID and vastly increased production of anti-viral IgG as compared to their single-cannulated counterparts. However, expression of B-cell survival factor BAFF was seen to be lower from day 8 onwards. Large, organised follicles were seen earlier than in primary immunisation with diffuse expression of CXCL13 at day 8. Conclusions Persistence of inflammation underlies many chronic inflammatory pathologies. Here we show that providing an excess of antigen at the peak of the inflammatory process appears to provoke a faster, more vigorous secondary immune response, both in terms of stromal cell activation and leucocyte recruitment, resulting in the avid production of anti-viral IgG antibodies. While this remains a spontaneously resolving process, it is the first step in characterising the secondary immune response in this novel murine model and brings us closer towards deciphering the factors triggering or preventing resolution. References Bombardieri M, Barone F, Lucchesi D, Nayar S, van den Berg WB, Proctor G et al. Inducible tertiary lymphoid structures, autoimmunity, and exocrine dysfunction in a novel model of salivary gland inflammation in C57...
BackgroundCostimulation via ICOS-ICOSL interaction is critical for germinal center reaction in secondary lymphoid organs, regulating T cell activation and maturation into cytokine-producing cells. In autoimmunity this signal has been extensively investigated and believed to contribute to the development of follicular T helper cells, autoreactive B cells and autoantibody production. Blocking ICOS-ICOSL interaction amelliorates disease in animal models of systemic lupus erythematosus and rheumatoid arthritis.ObjectivesTo dissect the role of ICOS-ICOSL pathway in a animal model of tertiary lymphiod organ (TLO) formation that mimics Sjögren's syndrome.MethodsSubmandibular glands of wild-type (wt), ICOSL, CD3ɛ and p55/75 (TNFR1) knockout (KO) mice were intra-ductally cannulated with luciferase-encoding replication-deficient adenovirus to induce TLO formation as previously described (1). A combination of immunofluorescence, quantitative RT-PCR, flow cytometry on digested salivary glands and in vitro mixed culture of lymphocytes and stroma were used.ResultsWe demonstrated that early post cannulation with adenovirus ICOSL is significantly upregulated within the salivary glands of infected mice on a population of activated stromal cells that express gp38, adhesion molecules, TNFR1 and TNFR2. In vivo experiments in ICOSLKO, CD3ɛKO and p55/75KO revealed a dramatic defect in TLO formation and reduced levels of ectopic lymphoid chemokines. ICOSL+ stromal cells stimulated effector T cells to produce lymphotoxin alpha (LTα) that in turn stimulates production of lymphoid chemokines by TNFR1+ stromal cells. These observations were further confirmed in vitro, in mixed co-cultures of lymphocytes, dendritic cells and stromal cells isolated from wt and ICOSLKO mice. This mechanism was further validated by inducing TLO formation in ICOSLKO reconstituted with wt bone marrow, that as predicted, showed a similar degree of impairment in the LTα/TNFR1 pathway as observed in the ICOSLKO mice.ConclusionsCollectively, these data demonstrate that ICOSL expression by stromal cells supports the up-regulation of LTα on T cells that in turn is required for chemokine expression via TNFR1 stimulation. These data provide a novel mechanism of action between ICOSL on stromal cells, ICOS on T cells and LTα pathways in the context of TLO formation.ReferencesBombardieri, Barone et al. JI. 2012Disclosure of InterestNone declared
Background Secondary lymphoid organs are characterized by concordance in time, function and response to stimuli between the lymphatic and vascular system that allows tight regulation of cellular recruitment and exit from the immune station (1,2). Defective drainage of the inflammatory cells from sites of chronic inflammation has been suggested to play a role in the sustenance of the inflammatory process. Objectives We used an inducible model of salivar gland ectopic lymphoneogenesis (3) in WT and KO mice, to evaluate whether similar events take place in the periphery and assess the modifications occurring to the vascular systems in different phases of the inflammatory process. Methods Flow cytometry and histological analysis in WT and LTβRKO salivary glands, cannulated with 10^8 p.f.u. of adenovirus, was performed at different time points post cannulation (pc). Flow cytometry was used to identify in digested tissues the CD45-EPCAM-CD31+GP38- blood endothelial cells (BEC) and CD45-EPCAM-CD31+GP38+ lymphatic endothelia cells (LEC). Immunofluorescence (IF) for CD31 and Lyve was used to validate the FACS data and establish the histological relationship between the lymphatic and the vascular system within the inflamed samples. Results By flow cytometry we observed a significant deflection in the percentage of blood endothelial cells in the early phases of the inflammatory process, followed by a progressive return to resting conditions during resolution. Conversely a significant increase in the number of lymphatic endothelial cells was observed in the early phase, followed by a significant decrease (p>0.01) at the peak of the inflammatory process and a drastic increase during resolution p= 0.01. Interestingly, while the first peak of lymphatic cells expansion corresponded to histological finding of a small number of lymphatic characterized by an enlarged lumen, the second peak was due to increase in the number of small lymphatics. This latest dramatic change in the number of the lymphatic vessels coincided with the infiltration of B-lymphocytes and peak of lymphoid cytokine lymphotoxin signal in the gland. Cannulation of LTβRKO, aimed to dissect the role of this molecule in lymphatic plasticity, demonstrated selective absence of the second peak of lymphatic proliferation and lack of lymphatic expansion by IF suggesting a key role for this molecule in lymphatic modification during resolution. Of note, LTβRKO mice did not show any significant difference as compared to the WT in the percentage of blood endothelial cells. Conclusions This study shows that ectopic lymphoid follicles associated vasculogenesis fails to recapitulate the tight regulation observed in secondary lymphoid organs. Of interest, the observed changes in the lymphatic bed seems to be regulated by the presence of ectopically expressed lymphocyte derived lymphotoxin, suggesting a novel therapeutic role for this molecule in inflammation. References Liao S, Ruddle NH. J Immunol. 2006. Tzeng TC., et al., J Immunol. 2010. Bombardieri M, Barone F,...
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