Saba Nayar

IL-22 regulates lymphoid chemokine production and assembly of tertiary lymphoid organs

Significance Ectopic clusters of immune cells that mimic the structure and function of secondary lymphoid organs are defined as tertiary lymphoid organs (TLOs). They have been observed at sites of chronic inflammation for decades, but their formation and function have remained enigmatic. TLOs are thought to contribute to disease pathogenesis by promoting autoreactive lymphocyte survival and autoantibody production. In this study we identify a novel role for the cytokine IL-22 in TLO development and biology. We provide evidence that IL-22 expression within TLOs is instrumental for the production of the lymphoid chemokines, chemokine (C-X-C motif) ligand 13 and chemokine (C-X-C motif) ligand 12, which in turn orchestrate B-cell clustering, lymphoid aggregation, and autoantibody production. Our data provide a strong rationale for targeting IL-22 in TLO-associated autoimmune diseases. The series of events leading to tertiary lymphoid organ (TLO) formation in mucosal organs following tissue damage remain unclear. Using a virus-induced model of autoantibody formation in the salivary glands of adult mice, we demonstrate that IL-22 provides a mechanistic link between mucosal infection, B-cell recruitment, and humoral autoimmunity. IL-22 receptor engagement is necessary and sufficient to promote differential expression of chemokine (C-X-C motif) ligand 12 and chemokine (C-X-C motif) ligand 13 in epithelial and fibroblastic stromal cells that, in turn, is pivotal for B-cell recruitment and organization of the TLOs. Accordingly, genetic and therapeutic blockade of IL-22 impairs and reverses TLO formation and autoantibody production. Our work highlights a critical role for IL-22 in TLO-induced pathology and provides a rationale for the use of IL-22–blocking agents in B-cell–mediated autoimmune conditions.

Inducible tertiary lymphoid structures, autoimmunity, and exocrine dysfunction in a novel model of salivary gland inflammation in C57BL/6 mice.

Salivary glands in patients with Sjögren’s syndrome (SS) develop ectopic lymphoid structures (ELS) characterized by B/T cell compartmentalization, the formation of high endothelial venules, follicular dendritic cell networks, functional B cell activation with expression of activation-induced cytidine deaminase, as well as local differentiation of autoreactive plasma cells. The mechanisms that trigger ELS formation, autoimmunity, and exocrine dysfunction in SS are largely unknown. In this article, we present a novel model of inducible ectopic lymphoid tissue formation, breach of humoral self-tolerance, and salivary hypofunction after delivery of a replication-deficient adenovirus-5 in submandibular glands of C57BL/6 mice through retrograde excretory duct cannulation. In this model, inflammation rapidly and consistently evolves from diffuse infiltration toward the development of SS-like periductal lymphoid aggregates within 2 wk from AdV delivery. These infiltrates progressively acquire ELS features and support functional GL7+/activation-induced cytidine deaminase+ germinal centers. Formation of ELS is preceded by ectopic expression of lymphoid chemokines CXCL13, CCL19, and lymphotoxin-β, and is associated with development of anti-nuclear Abs in up to 75% of mice. Finally, reduction in salivary flow was observed over 3 wk post-AdV infection, consistent with exocrine gland dysfunction as a consequence of the inflammatory response. This novel model has the potential to unravel the cellular and molecular mechanisms that regulate ELS formation and their role in exocrine dysfunction and autoimmunity in SS.

Stromal Cells in Chronic Inflammation and Tertiary Lymphoid Organ Formation

Inflammation is an unstable state. It either resolves or persists. Why inflammation persists and the factors that define tissue tropism remain obscure. Increasing evidence suggests that tissue-resident stromal cells not only provide positional memory but also actively regulate the differential accumulation of inflammatory cells within inflamed tissues. Furthermore, at many sites of chronic inflammation, structures that mimic secondary lymphoid tissues are observed, suggesting that chronic inflammation and lymphoid tissue formation share common activation programs. Similarly, blood and lymphatic endothelial cells contribute to tissue homeostasis and disease persistence in chronic inflammation. This review highlights our increasing understanding of the role of stromal cells in inflammation and summarizes the novel immunological role that stromal cells exert in the persistence of inflammatory diseases.

CLEC-2 is required for development and maintenance of lymph nodes.

The importance of CLEC-2, a natural ligand/receptor for Gp38/Podoplanin, in the formation of the lymphatic vasculature has recently been demonstrated. As the development and maintenance of lymph nodes (LNs) is dependent on the formation of the lymphatic vasculature and the differentiation of Gp38/Podoplanin(+) stromal cells, we investigated the role of CLEC-2 in lymphoneogenesis and LN homeostasis. Using constitutive Clec1b(-/-) mice, we showed that while CLEC-2 was not necessary for initiation of the LN anlage, it was required at late stages of development. Constitutive deletion of CLEC-2 induced a profound defect in lymphatic endothelial cell proliferation, resulting in lack of LNs at birth. In contrast, conditional deletion of CLEC-2 in the megakaryocyte/platelet lineage in Clec1b(fl/fl)PF4-Cre mice led to the development of blood-filled LNs and fibrosis, in absence of a proliferative defect of the lymphatic endothelial compartment. This phenotype was also observed in chimeric mice reconstituted with Clec1b(fl/fl)PF4-Cre bone marrow, indicating that CLEC-2 expression in platelets was required for LN integrity. We demonstrated that LNs of Clec1b(fl/fl)PF4-Cre mice are able to sustain primary immune responses but show a defect in immune cell recirculation after repeated immunizations, thus suggesting CLEC-2 as target in chronic immune response.

Homeostatic regulation of T cell trafficking by a B cell-derived peptide is impaired in autoimmune and chronic inflammatory disease

During an inflammatory response, lymphocyte recruitment into tissue must be tightly controlled because dysregulated trafficking contributes to the pathogenesis of chronic disease. Here we show that during inflammation and in response to adiponectin, B cells tonically inhibit T cell trafficking by secreting a peptide (PEPITEM) proteolytically derived from 14.3.3 zeta delta (14.3.3.ζδ) protein. PEPITEM binds cadherin-15 on endothelial cells, promoting synthesis and release of sphingosine-1 phosphate, which inhibits trafficking of T cells without affecting recruitment of other leukocytes. Expression of adiponectin receptors on B cells and adiponectin-induced PEPITEM secretion wanes with age, implying immune senescence of the pathway. Additionally, these changes are evident in individuals with type 1 diabetes or rheumatoid arthritis, and circulating PEPITEM in patient serum is reduced compared to that of healthy age-matched donors. In both diseases, tonic inhibition of T cell trafficking across inflamed endothelium is lost. Control of patient T cell trafficking is re-established by treatment with exogenous PEPITEM. Moreover, in animal models of peritonitis, hepatic ischemia-reperfusion injury, Salmonella infection, uveitis and Sjögrens syndrome, PEPITEM reduced T cell recruitment into inflamed tissues.

Inflammation-induced formation of fat-associated lymphoid clusters

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.

The expression of mouse CLEC-2 on leucocyte subsets varies according to their anatomical location and inflammatory state.

Expression of mouse C‐type lectin‐like receptor 2 (CLEC‐2) has been reported on circulating CD11bhigh Gr‐1high myeloid cells and dendritic cells (DCs) under basal conditions, as well as on a variety of leucocyte subsets following inflammatory stimuli or in vitro cell culture. However, previous studies assessing CLEC‐2 expression failed to use CLEC‐2‐deficient mice as negative controls and instead relied heavily on single antibody clones. Here, we generated CLEC‐2‐deficient adult mice using two independent approaches and employed two anti‐mouse CLEC‐2 antibody clones to investigate surface expression on hematopoietic cells from peripheral blood and secondary lymphoid organs. We rule out constitutive CLEC‐2 expression on resting DCs and show that CLEC‐2 is upregulated in response to LPS‐induced systemic inflammation in a small subset of activated DCs isolated from the mesenteric lymph nodes but not the spleen. Moreover, we demonstrate for the first time that peripheral blood B lymphocytes present exogenously derived CLEC‐2 and suggest that both circulating B lymphocytes and CD11bhigh Gr‐1high myeloid cells lose CLEC‐2 following entry into secondary lymphoid organs. These results have significant implications for our understanding of CLEC‐2 physiological functions

The role of non-hematopoietic stromal cells in the persistence of inflammation.

Inflammation results from the complex interaction between hematopoietic and stromal cells and growing evidence supports a key role for the stroma in driving the switch from acute resolving to persistence in chronic inflammatory diseases. Stromal cells have also been shown to play a critical role in cancer biology, being involved in cancer growth, dissemination, and inhibition of the autologous immune response, ultimately favoring persistence and metastatic spread. Similarly, blood and lymphatic endothelial cells contribute to tissue homeostasis during physiological inflammation but also lead to discorded leukocyte and tumor cell accumulation in pathological inflammation and cancer. This review aims to summarize the role that pathogenic stroma plays in the pathogenesis of diseases such as cancer and chronic inflammation.

Stromal Fibroblasts in Tertiary Lymphoid Structures: A Novel Target in Chronic Inflammation.

Tertiary lymphoid structures (TLS) are organized aggregates of lymphocytes, myeloid, and stromal cells that provide ectopic hubs for acquired immune responses. TLS share phenotypical and functional features with secondary lymphoid organs (SLO); however, they require persistent inflammatory signals to arise and are often observed at target sites of autoimmune disease, chronic infection, cancer, and organ transplantation. Over the past 10 years, important progress has been made in our understanding of the role of stromal fibroblasts in SLO development, organization, and function. A complex and stereotyped series of events regulate fibroblast differentiation from embryonic life in SLOs to lymphoid organ architecture observed in adults. In contrast, TLS-associated fibroblasts differentiate from postnatal, locally activated mesenchyme, predominantly in settings of inflammation and persistent antigen presentation. Therefore, there are critical differences in the cellular and molecular requirements that regulate SLO versus TLS development that ultimately impact on stromal and hematopoietic cell function. These differences may contribute to the pathogenic nature of TLS in the context of chronic inflammation and malignant transformation and offer a window of opportunity for therapeutic interventions in TLS associated pathologies.

Bimodal Expansion of the Lymphatic Vessels Is Regulated by the Sequential Expression of IL-7 and Lymphotoxin α1β2 in Newly Formed Tertiary Lymphoid Structures

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.