Epigenetic reader SP140 loss of function drives Crohn's disease due to uncontrolled macrophage topoisomerases.

How mis-regulated chromatin directly impacts human immune disorders is poorly understood. Speckled Protein 140 (SP140) is an immune-restricted PHD and bromodomain-containing epigenetic "reader," and SP140 loss-of-function mutations associate with Crohn's disease (CD), multiple sclerosis (MS), and chronic lymphocytic leukemia (CLL). However, the relevance of these mutations and mechanisms underlying SP140-driven pathogenicity remains unexplored. Using a global proteomic strategy, we identified SP140 as a repressor of topoisomerases (TOPs) that maintains heterochromatin and macrophage fate. In humans and mice, SP140 loss resulted in unleashed TOP activity, de-repression of developmentally silenced genes, and ultimately defective microbe-inducible macrophage transcriptional programs and bacterial killing that drive intestinal pathology. Pharmacological inhibition of TOP1/2 rescued these defects. Furthermore, exacerbated colitis was restored with TOP1/2 inhibitors in Sp140-/- mice, but not wild-type mice, in vivo. Collectively, we identify SP140 as a TOP repressor and reveal repurposing of TOP inhibition to reverse immune diseases driven by SP140 loss.

Engineered Sialylation of Pathogenic Antibodies In Vivo Attenuates Autoimmune Disease.

Self-reactive IgGs contribute to the pathology of autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. Paradoxically, IgGs are used to treat inflammatory diseases in the form of high-dose intravenous immunoglobulin (IVIG). Distinct glycoforms on the IgG crystallizable fragment (Fc) dictate these divergent functions. IgG anti-inflammatory activity is attributed to sialylation of the Fc glycan. We therefore sought to convert endogenous IgG to anti-inflammatory mediators in vivo by engineering solubilized glycosyltransferases that attach galactose or sialic acid. When both enzymes were administered in a prophylactic or therapeutic fashion, autoimmune inflammation was markedly attenuated in vivo. The enzymes worked through a similar pathway to IVIG, requiring DC-SIGN, STAT6 signaling, and FcγRIIB. Importantly, sialylation was highly specific to pathogenic IgG at the site of inflammation, driven by local platelet release of nucleotide-sugar donors. These results underscore the therapeutic potential of glycoengineering in vivo.

Sialylation of immunoglobulin E is a determinant of allergic pathogenicity.

Approximately one-third of the world's population suffers from allergies1. Exposure to allergens crosslinks immunoglobulin E (IgE) antibodies that are bound to mast cells and basophils, triggering the release of inflammatory mediators, including histamine2. Although IgE is absolutely required for allergies, it is not understood why total and allergen-specific IgE concentrations do not reproducibly correlate with allergic disease3-5. It is well-established that glycosylation of IgG dictates its effector function and has disease-specific patterns. However, whether IgE glycans differ in disease states or affect biological activity is completely unknown6. Here we perform an unbiased examination of glycosylation patterns of total IgE from individuals with a peanut allergy and from non-atopic individuals without allergies. Our analysis reveals an increase in sialic acid content on total IgE from individuals with a peanut allergy compared with non-atopic individuals. Removal of sialic acid from IgE attenuates effector-cell degranulation and anaphylaxis in several functional models of allergic disease. Therapeutic interventions-including removing sialic acid from cell-bound IgE with a neuraminidase enzyme targeted towards the IgE receptor FcεRI, and administering asialylated IgE-markedly reduce anaphylaxis. Together, these results establish IgE glycosylation, and specifically sialylation, as an important regulator of allergic disease.

Antibodies elicited in humans upon chimeric hemagglutinin-based influenza virus vaccination confer FcγR-dependent protection in vivo.

Antibody responses against highly conserved epitopes on the stalk domain of influenza virus hemagglutinin (HA) confer broad protection; however, such responses are limited. To effectively induce stalk-specific immunity against conserved HA epitopes, sequential immunization strategies have been developed based on chimeric HA (cHA) constructs featuring different head domains but the same stalk regions. Immunogenicity studies in small animal models, as well as in humans, revealed that cHA immunogens elicit stalk-specific IgG responses with broad specificity against heterologous influenza virus strains. However, the mechanisms by which these antibodies confer in vivo protection and the contribution of their Fc effector function remain unclear. To characterize the role of Fc-FcγR (Fcγ receptor) interactions to the in vivo protective activity of IgG antibodies elicited in participants in a phase I trial of a cHA vaccine candidate, we performed passive transfer studies of vaccine-elicited IgG antibodies in mice humanized for all classes of FcγRs, as well as in mice deficient for FcγRs. IgG antibodies elicited upon cHA vaccination completely protected FcγR humanized mice against lethal influenza virus challenge, while no protection was evident in FcγR-deficient mice, suggesting a major role for FcγR pathways in the protective function of vaccine-elicited IgG antibodies. These findings have important implications for influenza vaccine development, guiding the design of vaccination approaches with the capacity to elicit IgG responses with optimal Fc effector function.

Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas.

Dendritic cells (DCs), critical antigen-presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated monocyte-derived DCs (Mo-DCs) develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram-negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen-presenting function, Mo-DCs are as active as classical DCs, including cross-presentation of proteins and live gram-negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN(+) cells with critical functions of DCs.

Modulating T Follicular Cells In Vivo Enhances Antigen-Specific Humoral Immunity.

Generation of high-affinity IgG is essential for defense against infections and cancer, which is the intended consequence of many vaccines, but can cause autoimmune and inflammatory diseases when inappropriately directed against self. The interplay of T follicular helper (TFH) cells and T follicular regulatory (TFR) cells is critical for the production of high-affinity IgG of a specific subclass. In this study, we sought to improve Ag-specific IgG responses with two interventions intended to transiently diminish TFR cell influence. First, adult mice were administered an antibiotic mixture (ABX) for an extended period to deplete the immunoregulatory intestinal microbiota. This intriguingly increased TFH cell and reduced TFR cell numbers. 2,4,6-Trinitrophenyl hapten conjugated to keyhole limpet hemocyanin immunization resulted in higher affinity 2,4,6-trinitrophenyl hapten-specific IgG1 in ABX mice compared with controls. In a model of IgG-driven inflammatory nephritis, ABX mice had significantly worse nephritis accompanied by higher affinity Ag-specific IgG2b and enriched TFH cells compared with controls. Second, we sought to functionally manipulate TFH and TFR cells, which both express the checkpoint inhibitory molecule, PD-1, by administration of anti-PD-1 during immunization. This intervention enhanced the affinity of Ag-specific IgG of the appropriate subclass and increased in TFH cells following 2,4,6-trinitrophenyl hapten conjugated to keyhole limpet hemocyanin immunization and nephritis induction. These results suggest that altering TFH and TFR cell ratios during immunization is an appealing strategy to qualitatively improve Ag- and subclass-specific IgG responses.

IgE Glycosylation in Health and Disease.

IgE are absolutely required for initiation of allergy reactions, which affect over 20% of the world's population. IgE are the least prevalent immunoglobulins in circulation with 12-h and 2-day half-lives in mouse and human serum, respectively, but an extended tissue half-life of 3-weeks bound to the surface of mast cells by the high affinity IgE receptor, FcεRI (Gould and Sutton 2008). Although the importance of glycosylation to IgG biology is well established, less is known regarding the contribution of IgE glycosylation to allergic inflammation. IgE has seven and nine N-linked glycosylation sites distributed across human and murine constant chains, respectively. Here we discuss studies that have analyzed IgE glycosylation and its function, and how IgE glycosylation contributions to health and disease.

Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway.

High-dose intravenous immunoglobulin is a widely used therapeutic preparation of highly purified immunoglobulin G (IgG) antibodies. It is administered at high doses (1-2 grams per kilogram) for the suppression of autoantibody-triggered inflammation in a variety of clinical settings. This anti-inflammatory activity of intravenous immunoglobulin is triggered by a minor population of IgG crystallizable fragments (Fcs), with glycans terminating in α2,6 sialic acids (sFc) that target myeloid regulatory cells expressing the lectin dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN; also known as CD209). Here, to characterize this response in detail, we generated humanized DC-SIGN mice (hDC-SIGN), and demonstrate that the anti-inflammatory activity of intravenous immunoglobulin can be recapitulated by the transfer of bone-marrow-derived sFc-treated hDC-SIGN(+) macrophages or dendritic cells into naive recipients. Furthermore, sFc administration results in the production of IL-33, which, in turn, induces expansion of IL-4-producing basophils that promote increased expression of the inhibitory Fc receptor FcγRIIB on effector macrophages. Systemic administration of the T(H)2 cytokines IL-33 or IL-4 upregulates FcγRIIB on macrophages, and suppresses serum-induced arthritis. Consistent with these results, transfer of IL-33-treated basophils suppressed induced arthritic inflammation. This novel DC-SIGN-T(H)2 pathway initiated by an endogenous ligand, sFc, provides an intrinsic mechanism for maintaining immune homeostasis that could be manipulated to provide therapeutic benefit in autoimmune diseases.

Acute inflammation primes myeloid effector cells for anti-inflammatory STAT6 signaling.

The anti-inflammatory drug high-dose intravenous immunoglobulin, widely used to suppress inflammation, depends on a specific α-2,6-sialylated glycoform of IgG Fc to induce Interleukin 4 (IL-4) and Signal Transducer and Activator of Transcription 6 (STAT6) signaling for its activity. Here we show that anti-inflammatory activities of IL-4 can be attributed to the direct action of this cytokine on myeloid effector cells, depending on their expression of the IL-4 receptor alpha chain (IL-4Rα/CD124). However, in their basal state, these cells express low levels of IL-4Rα and would not be expected to result in significant signaling compared with other cell populations. This apparent paradox can be explained by the observation that during inflammation, triggered by a variety of stimuli (including autoantibodies, adjuvants, and TLR ligands), IL-4Rα is up-regulated specifically on these cells, priming them for STAT6 signaling. The regulation is mediated by a soluble, proteinase K-sensitive factor, released to the circulation by bone marrow-derived, non-B/non-T cells found in several organs, including the lungs, and fat. We propose that this regulation is part of a homeostatic mechanism to limit excessive inflammation and tissue damage. High-dose intravenous immunoglobulin thus exploits an endogenous feedback loop, general to inflammation, that could be further targeted for therapeutic purposes.

Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals.

Antibodies to conserved epitopes on the human immunodeficiency virus (HIV) surface protein gp140 can protect against infection in non-human primates, and some infected individuals show high titres of broadly neutralizing immunoglobulin (Ig)G antibodies in their serum. However, little is known about the specificity and activity of these antibodies. To characterize the memory antibody responses to HIV, we cloned 502 antibodies from HIV envelope-binding memory B cells from six HIV-infected patients with broadly neutralizing antibodies and low to intermediate viral loads. We show that in these patients, the B-cell memory response to gp140 is composed of up to 50 independent clones expressing high affinity neutralizing antibodies to the gp120 variable loops, the CD4-binding site, the co-receptor-binding site, and to a new neutralizing epitope that is in the same region of gp120 as the CD4-binding site. Thus, the IgG memory B-cell compartment in the selected group of patients with broad serum neutralizing activity to HIV is comprised of multiple clonal responses with neutralizing activity directed against several epitopes on gp120.

An engineered immunomodulatory IgG1 Fc suppresses autoimmune inflammation through pathways shared with i.v. immunoglobulin.

Immunoglobulin G (IgG) antibodies in the form of high-dose intravenous immunoglobulin (IVIG) exert immunomodulatory activity and are used in this capacity to treat inflammatory and autoimmune diseases. Reductionist approaches have revealed that terminal sialylation of the single asparagine-linked (N-linked) glycan at position 297 of the IgG1 Fc bestows antiinflammatory activity, which can be recapitulated by introduction of an F241A point mutation in the IgG1 Fc (FcF241A). Here, we examined the antiinflammatory activity of CHO-K1 cell-produced FcF241A in vivo in models of autoimmune inflammation and found it to be independent of sialylation. Intriguingly, sialylation markedly improved the half-life and bioavailability of FcF241A via impaired interaction with the asialoglycoprotein receptor ASGPR. Further, FcF241A suppressed inflammation through the same molecular pathways as IVIG and sialylated IgG1 Fc and required the C-type lectin SIGN-R1 in vivo. This contrasted with FcAbdeg (efgartigimod), an engineered IgG1 Fc with enhanced neonatal Fc receptor (FcRn) binding, which reduced total serum IgG concentrations, independent of SIGN-R1. When coadministered, FcF241A and FcAbdeg exhibited combinatorial antiinflammatory activity. Together, these results demonstrated that the antiinflammatory activity of FcF241A requires SIGN-R1, similarly to that of high-dose IVIG and sialylated IgG1, and can be used in combination with other antiinflammatory therapeutics that rely on divergent pathways, including FcAbdeg.

Memory T(H)2 cells induce alternatively activated macrophages to mediate protection against nematode parasites.

Although primary and memory responses against bacteria and viruses have been studied extensively, T helper type 2 (T(H)2) effector mechanisms leading to host protection against helminthic parasites remain elusive. Examination of the intestinal epithelial submucosa of mice after primary and secondary infections by a natural gastrointestinal parasite revealed a distinct immune-cell infiltrate after challenge, featuring interleukin-4-expressing memory CD4(+) T cells that induced IL-4 receptor(hi) (IL-4R(hi)) CD206(+) alternatively activated macrophages. In turn, these alternatively activated macrophages (AAMacs) functioned as important effector cells of the protective memory response contributing to parasite elimination, demonstrating a previously unknown mechanism for host protection against intestinal helminths.

Reaching macs-imum potential.

Single-cell profiling of prenatal samples reveals multiple macrophage types and states, including microglia-like cells in non-neuronal tissues.

Lung injury induces a polarized immune response by self antigen-specific Foxp3 + regulatory T cells.

Self antigen-specific T cells are prevalent in the mature adaptive immune system, but are regulated through multiple mechanisms of tolerance. However, inflammatory conditions such as tissue injury may provide these T cells with an opportunity to break tolerance and trigger autoimmunity. To understand how the T cell repertoire responds to the presentation of self antigen under highly stimulatory conditions, we used peptide:MHCII tetramers to track the behavior of endogenous CD4 + T cells with specificity to a lung-expressed self antigen in mouse models of immune-mediated lung injury. Acute injury resulted in the exclusive expansion of regulatory T cells (Tregs) that was dependent on self antigen recognition and IL-2. Conversely, conventional T cells of the same self antigen specificity remained unresponsive, even following Treg ablation. Thus, the self antigen-specific T cell repertoire is poised to serve a regulatory function during acute tissue damage to limit further damage and the possibility of autoimmunity.

Lung injury induces a polarized immune response by self-antigen-specific CD4+ Foxp3+ regulatory T cells.

Self-antigen-specific T cells are prevalent in the mature adaptive immune system but are regulated through multiple mechanisms of tolerance. However, inflammatory conditions such as tissue injury may allow these T cells to break tolerance and trigger autoimmunity. To understand how the T cell repertoire responds to the presentation of self-antigen under highly stimulatory conditions, we use peptide:major histocompatibility complex (MHC) class II tetramers to track the behavior of endogenous CD4+ T cells with specificity to a lung-expressed self-antigen in mouse models of immune-mediated lung injury. Acute injury results in the exclusive expansion of CD4+ regulatory T cells (Tregs) that is dependent on self-antigen recognition and interleukin-2 (IL-2). Conversely, conventional CD4+ T cells of the same self-antigen specificity remain unresponsive even following Treg ablation. Thus, the self-antigen-specific CD4+ T cell repertoire is poised to serve a regulatory function during acute tissue damage to limit further damage and the possibility of autoimmunity.

Microbiota-dependent indole production is required for the development of collagen-induced arthritis.

Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model we identify alterations in tryptophan metabolism, and specifically indole, that correlate with disease. We demonstrate that both bacteria and dietary tryptophan are required for disease, and indole supplementation is sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1ß; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colon lymphocytes to indole increased expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a novel therapeutic pathway for RA and SpA.

Microbiota-dependent indole production stimulates the development of collagen-induced arthritis in mice.

Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model, we identified alterations in tryptophan metabolism, and specifically indole, that correlated with disease. We demonstrated that both bacteria and dietary tryptophan were required for disease and that indole supplementation was sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1ß; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colonic lymphocytes to indole increased the expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a unique therapeutic pathway for RA and SpA.

Sialylation as an Important Regulator of Antibody Function.

Antibodies play a critical role in linking the adaptive immune response to the innate immune system. In humans, antibodies are categorized into five classes, IgG, IgM, IgA, IgE, and IgD, based on constant region sequence, structure, and tropism. In serum, IgG is the most abundant antibody, comprising 75% of antibodies in circulation, followed by IgA at 15%, IgM at 10%, and IgD and IgE are the least abundant. All human antibody classes are post-translationally modified by sugars. The resulting glycans take on many divergent structures and can be attached in an N-linked or O-linked manner, and are distinct by antibody class, and by position on each antibody. Many of these glycan structures on antibodies are capped by sialic acid. It is well established that the composition of the N-linked glycans on IgG exert a profound influence on its effector functions. However, recent studies have described the influence of glycans, particularly sialic acid for other antibody classes. Here, we discuss the role of glycosylation, with a focus on terminal sialylation, in the biology and function across all antibody classes. Sialylation has been shown to influence not only IgG, but IgE, IgM, and IgA biology, making it an important and unappreciated regulator of antibody function.

Identification of a receptor required for the anti-inflammatory activity of IVIG.

The anti-inflammatory activity of intravenous Ig (IVIG) results from a minor population of the pooled IgG molecules that contains terminal alpha2,6-sialic acid linkages on their Fc-linked glycans. These anti-inflammatory properties can be recapitulated with a fully recombinant preparation of appropriately sialylated IgG Fc fragments. We now demonstrate that these sialylated Fcs require a specific C-type lectin, SIGN-R1, (specific ICAM-3 grabbing non-integrin-related 1) expressed on macrophages in the splenic marginal zone. Splenectomy, loss of SIGN-R1(+) cells in the splenic marginal zone, blockade of the carbohydrate recognition domain (CRD) of SIGN-R1, or genetic deletion of SIGN-R1 abrogated the anti-inflammatory activity of IVIG or sialylated Fc fragments. Although SIGN-R1 has not previously been shown to bind to sialylated glycans, we demonstrate that it preferentially binds to 2,6-sialylated Fc compared with similarly sialylated, biantennary glycoproteins, thus suggesting that a specific binding site is created by the sialylation of IgG Fc. A human orthologue of SIGN-R1, DC-SIGN, displays a similar binding specificity to SIGN-R1 but differs in its cellular distribution, potentially accounting for some of the species differences observed in IVIG protection. These studies thus identify an antibody receptor specific for sialylated Fc, and present the initial step that is triggered by IVIG to suppress inflammation.