Serum Amyloid A Proteins Induce Pathogenic Th17 Cells and Promote Inflammatory Disease.

Lymphoid cells that produce interleukin (IL)-17 cytokines protect barrier tissues from pathogenic microbes but are also prominent effectors of inflammation and autoimmune disease. T helper 17 (Th17) cells, defined by RORγt-dependent production of IL-17A and IL-17F, exert homeostatic functions in the gut upon microbiota-directed differentiation from naive CD4+ T cells. In the non-pathogenic setting, their cytokine production is regulated by serum amyloid A proteins (SAA1 and SAA2) secreted by adjacent intestinal epithelial cells. However, Th17 cell behaviors vary markedly according to their environment. Here, we show that SAAs additionally direct a pathogenic pro-inflammatory Th17 cell differentiation program, acting directly on T cells in collaboration with STAT3-activating cytokines. Using loss- and gain-of-function mouse models, we show that SAA1, SAA2, and SAA3 have distinct systemic and local functions in promoting Th17-mediated inflammatory diseases. These studies suggest that T cell signaling pathways modulated by the SAAs may be attractive targets for anti-inflammatory therapies.

Transcription factor RORα enforces stability of the Th17 cell effector program by binding to a Rorc cis-regulatory element.

T helper 17 (Th17) cells regulate mucosal barrier defenses but also promote multiple autoinflammatory diseases. Although many molecular determinants of Th17 cell differentiation have been elucidated, the transcriptional programs that sustain Th17 cells in vivo remain obscure. The transcription factor RORγt is critical for Th17 cell differentiation; however, it is not clear whether the closely related RORα, which is co-expressed in Th17 cells, has a distinct role. Here, we demonstrated that although dispensable for Th17 cell differentiation, RORα was necessary for optimal Th17 responses in peripheral tissues. The absence of RORα in T cells led to reductions in both RORγt expression and effector function among Th17 cells. Cooperative binding of RORα and RORγt to a previously unidentified Rorc cis-regulatory element was essential for Th17 lineage maintenance in vivo. These data point to a non-redundant role of RORα in Th17 lineage maintenance via reinforcement of the RORγt transcriptional program.

An IL-23R/IL-22 Circuit Regulates Epithelial Serum Amyloid A to Promote Local Effector Th17 Responses.

RORγt(+) Th17 cells are important for mucosal defenses but also contribute to autoimmune disease. They accumulate in the intestine in response to microbiota and produce IL-17 cytokines. Segmented filamentous bacteria (SFB) are Th17-inducing commensals that potentiate autoimmunity in mice. RORγt(+) T cells were induced in mesenteric lymph nodes early after SFB colonization and distributed across different segments of the gastrointestinal tract. However, robust IL-17A production was restricted to the ileum, where SFB makes direct contact with the epithelium and induces serum amyloid A proteins 1 and 2 (SAA1/2), which promote local IL-17A expression in RORγt(+) T cells. We identified an SFB-dependent role of type 3 innate lymphoid cells (ILC3), which secreted IL-22 that induced epithelial SAA production in a Stat3-dependent manner. This highlights the critical role of tissue microenvironment in activating effector functions of committed Th17 cells, which may have important implications for how these cells contribute to inflammatory disease.

ETV5 promotes lupus pathogenesis and follicular helper T cell differentiation by inducing osteopontin expression.

Follicular helper T (TFH) cells mediate germinal center reactions to generate high affinity antibodies against specific pathogens, and their excessive production is associated with the pathogenesis of systemic autoimmune diseases such as systemic lupus erythematosus (SLE). ETV5, a member of the ETS transcription factor family, promotes TFH cell differentiation in mice. In this study, we examined the role of ETV5 in the pathogenesis of lupus in mice and humans. T cell-specific deletion of Etv5 alleles ameliorated TFH cell differentiation and autoimmune phenotypes in lupus mouse models. Further, we identified SPP1 as an ETV5 target that promotes TFH cell differentiation in both mice and humans. Notably, extracellular osteopontin (OPN) encoded by SPP1 enhances TFH cell differentiation by activating the CD44-AKT signaling pathway. Furthermore, ETV5 and SPP1 levels were increased in CD4+ T cells from patients with SLE and were positively correlated with disease activity. Taken together, our findings demonstrate that ETV5 is a lupus-promoting transcription factor, and secreted OPN promotes TFH cell differentiation.

Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells.

Cell-mediated immunity critically depends on the localization of lymphocytes at sites of infection. While some memory T cells recirculate, a distinct lineage (resident memory T cells (T(RM) cells)) are embedded in nonlymphoid tissues (NLTs) and mediate potent protective immunity. However, the defining transcriptional basis for the establishment of T(RM) cells is unknown. We found that CD8(+) T(RM) cells lacked expression of the transcription factor KLF2 and its target gene S1pr1 (which encodes S1P1, a receptor for sphingosine 1-phosphate). Forced expression of S1P1 prevented the establishment of T(RM) cells. Cytokines that induced a T(RM) cell phenotype (including transforming growth factor-ß (TGF-ß), interleukin 33 (IL-33) and tumor-necrosis factor) elicited downregulation of KLF2 expression in a pathway dependent on phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt, which suggested environmental regulation. Hence, regulation of KLF2 and S1P1 provides a switch that dictates whether CD8(+) T cells commit to recirculating or tissue-resident memory populations.

Distinct Roles of Brd2 and Brd4 in Potentiating the Transcriptional Program for Th17 Cell Differentiation.

The BET proteins are major transcriptional regulators and have emerged as new drug targets, but their functional distinction has remained elusive. In this study, we report that the BET family members Brd2 and Brd4 exert distinct genomic functions at genes whose transcription they co-regulate during mouse T helper 17 (Th17) cell differentiation. Brd2 is associated with the chromatin insulator CTCF and the cohesin complex to support cis-regulatory enhancer assembly for gene transcriptional activation. In this context, Brd2 binds the transcription factor Stat3 in an acetylation-sensitive manner and facilitates Stat3 recruitment to active enhancers occupied with transcription factors Irf4 and Batf. In parallel, Brd4 temporally controls RNA polymerase II (Pol II) processivity during transcription elongation through cyclin T1 and Cdk9 recruitment and Pol II Ser2 phosphorylation. Collectively, our study uncovers both separate and interdependent Brd2 and Brd4 functions in potentiating the genetic program required for Th17 cell development and adaptive immunity.

The transcription factor KLF2 restrains CD4⁺ T follicular helper cell differentiation.

T follicular helper (Tfh) cells are essential for efficient B cell responses, yet the factors that regulate differentiation of this CD4(+) T cell subset are incompletely understood. Here we found that the KLF2 transcription factor serves to restrain Tfh cell generation. Induced KLF2 deficiency in activated CD4(+) T cells led to increased Tfh cell generation and B cell priming, whereas KLF2 overexpression prevented Tfh cell production. KLF2 promotes expression of the trafficking receptor S1PR1, and S1PR1 downregulation is essential for efficient Tfh cell production. However, KLF2 also induced expression of the transcription factor Blimp-1, which repressed transcription factor Bcl-6 and thereby impaired Tfh cell differentiation. Furthermore, KLF2 induced expression of the transcription factors T-bet and GATA3 and enhanced Th1 differentiation. Hence, our data indicate KLF2 is pivotal for coordinating CD4(+) T cell differentiation through two distinct and complementary mechanisms: via control of T cell localization and by regulation of lineage-defining transcription factors.

Leveraging chromatin accessibility for transcriptional regulatory network inference in T Helper 17 Cells.

Transcriptional regulatory networks (TRNs) provide insight into cellular behavior by describing interactions between transcription factors (TFs) and their gene targets. The assay for transposase-accessible chromatin (ATAC)-seq, coupled with TF motif analysis, provides indirect evidence of chromatin binding for hundreds of TFs genome-wide. Here, we propose methods for TRN inference in a mammalian setting, using ATAC-seq data to improve gene expression modeling. We test our methods in the context of T Helper Cell Type 17 (Th17) differentiation, generating new ATAC-seq data to complement existing Th17 genomic resources. In this resource-rich mammalian setting, our extensive benchmarking provides quantitative, genome-scale evaluation of TRN inference, combining ATAC-seq and RNA-seq data. We refine and extend our previous Th17 TRN, using our new TRN inference methods to integrate all Th17 data (gene expression, ATAC-seq, TF knockouts, and ChIP-seq). We highlight newly discovered roles for individual TFs and groups of TFs ("TF-TF modules") in Th17 gene regulation. Given the popularity of ATAC-seq, which provides high-resolution with low sample input requirements, we anticipate that our methods will improve TRN inference in new mammalian systems, especially in vivo, for cells directly from humans and animal models.

Characterization of a Major Allergen from Mongolian Oak, Quercus mongolica, a Dominant Species of Oak in Korea.

BACKGROUND: Oaks are the most common trees in Korean forests, and Mongolian oak, Quercus mongolica, is the dominant species. However, no allergen has been characterized from Mongolian oak. In this study, we tried to characterize a major allergen from Mongolian oak. METHODS: A molecule homologous to pathogenesis-related 10 (PR-10)-like protein, Que m 1, was cloned by RT-PCR. Its recombinant protein, along with Que a 1, an allergen from white oak (Q. alba), was produced. The allergenicity and diagnostic value of recombinant Que m 1, Que a 1, and Bet v 1 proteins were compared by ELISA using sera from oak-sensitized subjects. A basophil activation test was also performed using CD63 expression as an activation marker. RESULTS: Que m 1 sequence shares 57.5-96.2% amino acid sequence identity with PR-10-like allergens from various plants. Specific IgE to recombinant Que m 1, Que a 1, and Bet v 1 were detected in 92.0, 74.0, and 38.0% of 50 serum samples from Korean tree pollinosis patients. Recombinant Que m 1 was able to inhibit IgE reactivity to Que a 1 and Bet v 1, indicating its strong cross-reactivity. The activation patterns of basophils from 5 patients were similar in terms of the CD63 expression and protein concentration of challenged Bet v 1 and Que m 1. CONCLUSIONS: A major allergen, Que m 1, was cloned, and its recombinant protein was produced from Mongolian oak, a dominant species in Korea. Recombinant Que m 1 is potentially useful for the diagnosis and treatment of tree pollinosis in Korea.

Virtual memory CD8 T cells display unique functional properties.

Previous studies revealed the existence of foreign antigen-specific memory phenotype CD8 T cells in unimmunized mice. Considerable evidence suggests this population, termed "virtual memory" (VM) CD8 T cells, arise via physiological homeostatic mechanisms. However, the antigen-specific function of VM cells is poorly characterized, and hence their potential contribution to immune responses against pathogens is unclear. Here we show that naturally occurring, polyclonal VM cells have unique functional properties, distinct from either naïve or antigen-primed memory CD8 T cells. In striking contrast to conventional memory cells, VM cells showed poor T cell receptor-induced IFN-γ synthesis and preferentially differentiated into central memory phenotype cells after priming. Importantly, VM cells showed efficient control of Listeria monocytogenes infection, indicating memory-like capacity to eliminate certain pathogens. These data suggest naturally arising VM cells display unique functional traits, allowing them to form a bridge between the innate and adaptive phase of a response to pathogens.

Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori.

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.

Derivation and maintenance of virtual memory CD8 T cells.

Memory CD8(+) T cells are an important component of the adaptive immune response against many infections, and understanding how Ag-specific memory CD8(+) T cells are generated and maintained is crucial for the development of vaccines. We recently reported the existence of memory-phenotype, Ag-specific CD8(+) T cells in unimmunized mice (virtual memory or VM cells). However, it was not clear when and where these cells are generated during normal development, nor the factors required for their production and maintenance. This issue is especially pertinent given recent data showing that memory-like CD8 T cells can be generated in the thymus, in a bystander response to IL-4. In this study, we show that the size of the VM population is reduced in IL-4R-deficient animals. However, the VM population appears first in the periphery and not the thymus of normal animals, suggesting this role of IL-4 is manifest following thymic egress. We also show that the VM pool is durable, showing basal proliferation and long-term maintenance in normal animals, and also being retained during responses to unrelated infection.

5-aminosalicylic acid suppresses osteoarthritis through the OSCAR-PPARγ axis.

Osteoarthritis (OA) is a progressive and irreversible degenerative joint disease that is characterized by cartilage destruction, osteophyte formation, subchondral bone remodeling, and synovitis. Despite affecting millions of patients, effective and safe disease-modifying osteoarthritis drugs are lacking. Here we reveal an unexpected role for the small molecule 5-aminosalicylic acid (5-ASA), which is used as an anti-inflammatory drug in ulcerative colitis. We show that 5-ASA competes with extracellular-matrix collagen-II to bind to osteoclast-associated receptor (OSCAR) on chondrocytes. Intra-articular 5-ASA injections ameliorate OA generated by surgery-induced medial-meniscus destabilization in male mice. Significantly, this effect is also observed when 5-ASA was administered well after OA onset. Moreover, mice with DMM-induced OA that are treated with 5-ASA at weeks 8-11 and sacrificed at week 12 have thicker cartilage than untreated mice that were sacrificed at week 8. Mechanistically, 5-ASA reverses OSCAR-mediated transcriptional repression of PPARγ in articular chondrocytes, thereby suppressing COX-2-related inflammation. It also improves chondrogenesis, strongly downregulates ECM catabolism, and promotes ECM anabolism. Our results suggest that 5-ASA could serve as a DMOAD.

Transcriptional and Epigenetic Regulation of Context-Dependent Plasticity in T-Helper Lineages.

Th cell lineage determination and functional specialization are tightly linked to the activation of lineage-determining transcription factors (TFs) that bind cis-regulatory elements. These lineage-determining TFs act in concert with multiple layers of transcriptional regulators to alter the epigenetic landscape, including DNA methylation, histone modification and three-dimensional chromosome architecture, in order to facilitate the specific Th gene expression programs that allow for phenotypic diversification. Accumulating evidence indicates that Th cell differentiation is not as rigid as classically held; rather, extensive phenotypic plasticity is an inherent feature of T cell lineages. Recent studies have begun to uncover the epigenetic programs that mechanistically govern T cell subset specification and immunological memory. Advances in next generation sequencing technologies have allowed global transcriptomic and epigenomic interrogation of CD4+ Th cells that extends previous findings focusing on individual loci. In this review, we provide an overview of recent genome-wide insights into the transcriptional and epigenetic regulation of CD4+ T cell-mediated adaptive immunity and discuss the implications for disease as well as immunotherapies.

IgSF11 deficiency alleviates osteoarthritis in mice by suppressing early subchondral bone changes.

Osteoarthritis (OA) is a degenerative joint disease. While it is classically characterized by articular cartilage destruction, OA affects all tissues in the joints and is thus also accompanied by local inflammation, subchondral bone changes, and persistent pain. However, our understanding of the underlying subchondral bone dynamics during OA progression is poor. Here, we demonstrate the contribution of immunoglobulin superfamily 11 (IgSF11) to OA subchondral bone remodeling by using a murine model. In particular, IgSF11 was quickly expressed by differentiating osteoclasts and upregulated in subchondral bone soon after destabilization-of-the-medial-meniscus (DMM)-induced OA. In mice, IgSF11 deficiency not only suppressed subchondral bone changes in OA but also blocked cartilage destruction. The IgSF11-expressing cells in OA subchondral bone were found to be involved in osteoclast maturation and bone resorption and colocalized with receptor-activator of nuclear-factor κ-B (RANK), the key osteoclast differentiation factor. Thus, our study shows that blocking early subchondral bone changes in OA can ameliorate articular cartilage destruction in OA.

Serum amyloid A-dependent inflammasome activation and acute injury in a mouse model of experimental stroke.

Serum amyloid A (SAA) proteins increase dramatically in the blood following inflammation. Recently, SAAs are increased in humans following stroke and in ischemic animal models. However, the impact of SAAs on whether this signal is critical in the ischemic brain remains unknown. Therefore, we investigated the role of SAA and SAA signaling in the ischemic brain. Wildtype and SAA deficient mice were exposed to middle cerebral artery occlusion and reperfusion, examined for the impact of infarct volumes, behavioral changes, inflammatory markers, TUNEL staining, and BBB changes. The underlying mechanisms were investigated using SAA deficient mice, transgenic mice and viral vectors. SAA levels were significantly increase following MCAo and mice deficient in SAAs showed reduced infarct volumes and improved behavioral outcomes. SAA deficient mice showed a reduction in TUNEL staining, inflammation and decreased glial activation. Mice lacking acute phase SAAs demonstrated a reduction in expression of the NLRP3 inflammasome and SAA/NLRP3 KO mice showed improvement. Restoration of SAA expression via SAA tg mice or adenoviral expression reestablished the detrimental effects of SAA. A reduction in BBB permeability was seen in the SAA KO mice and anti-SAA antibody treatment reduced the effects on ischemic injury. SAA signaling plays a critical role in regulating NLRP3-induced inflammation and glial activation in the ischemic brain. Blocking this signal will be a promising approach for treating ischemic stroke.

Context-Dependent Regulation of Type17 Immunity by Microbiota at the Intestinal Barrier.

T-helper-17 (Th17) cells and related IL-17-producing (type17) lymphocytes are abundant at the epithelial barrier. In response to bacterial and fungal infection, the signature cytokines IL-17A/F and IL-22 mediate the antimicrobial immune response and contribute to wound healing of injured tissues. Despite their protective function, type17 lymphocytes are also responsible for various chronic inflammatory disorders, including inflammatory bowel disease (IBD) and colitis associated cancer (CAC). A deeper understanding of type17 regulatory mechanisms could ultimately lead to the discovery of therapeutic strategies for the treatment of chronic inflammatory disorders and the prevention of cancer. In this review, we discuss the current understanding of the development and function of type17 immune cells at the intestinal barrier, focusing on the impact of microbiota-immune interactions on intestinal barrier homeostasis and disease etiology.

TREGking From Gut to Brain: The Control of Regulatory T Cells Along the Gut-Brain Axis.

The human gastrointestinal tract has an enormous and diverse microbial community, termed microbiota, that is necessary for the development of the immune system and tissue homeostasis. In contrast, microbial dysbiosis is associated with various inflammatory and autoimmune diseases as well as neurological disorders in humans by affecting not only the immune system in the gastrointestinal tract but also other distal organs. FOXP3+ regulatory T cells (Tregs) are a subset of CD4+ helper T cell lineages that function as a gatekeeper for immune activation and are essential for peripheral autoimmunity prevention. Tregs are crucial to the maintenance of immunological homeostasis and tolerance at barrier regions. Tregs reside in both lymphoid and non-lymphoid tissues, and tissue-resident Tregs have unique tissue-specific phenotype and distinct function. The gut microbiota has an impact on Tregs development, accumulation, and function in periphery. Tregs, in turn, modulate antigen-specific responses aimed towards gut microbes, which supports the host-microbiota symbiotic interaction in the gut. Recent studies have indicated that Tregs interact with a variety of resident cells in central nervous system (CNS) to limit the progression of neurological illnesses such as ischemic stroke, Alzheimer's disease, and Parkinson's disease. The gastrointestinal tract and CNS are functionally connected, and current findings provide insights that Tregs function along the gut-brain axis by interacting with immune, epithelial, and neuronal cells. The purpose of this study is to explain our current knowledge of the biological role of tissue-resident Tregs, as well as the interaction along the gut-brain axis.