Resident Synovial Macrophages in Synovial Fluid: Implications for Immunoregulation in Infectious and Inflammatory Arthritis.

Objectives: Resident synovial macrophages (RSM) provide immune sequestration of the joint space and are likely involved in initiation and perpetuation of the joint-specific immune response. We sought to identify RSM in synovial fluid (SF) and demonstrate migratory ability, in additional to functional changes that may perpetuate a chronic inflammatory response within joint spaces. Methods: We recruited human patients presenting with undifferentiated arthritis in multiple clinical settings. We used flow cytometry to identify mononuclear cells in peripheral blood and SF. We used a novel transwell migration assay with human ex-vivo synovium obtained intra-operatively to validate flow cytometry findings. We used single cell RNA-sequencing (scRNA-seq) to further identify macrophage/monocyte subsets. ELISA was used to evaluate the bone-resorption potential of SF. Results: We were able to identify a rare population of CD14dim, OPG+, ZO-1+ cells consistent with RSM in SF via flow cytometry. These cells were relatively enriched in the SF during infectious processes, but absolutely decreased compared to healthy controls. Similar putative RSM were identified using ex vivo migration assays when MCP-1 and LPS were used as migratory stimulus. scRNA-seq revealed a population consistent with RSM transcriptionally related to CD56+ cytotoxic dendritic cells and IDO+ M2 macrophages. Conclusion: We identified a rare cell population consistent with RSM, indicating these cells are likely migratory and able to initiate or coordinate both acute (septic) or chronic (autoimmune or inflammatory) arthritis. RSM analysis via scRNA-seq indicated these cells are M2 skewed, capable of antigen presentation, and have consistent functions in both septic and inflammatory arthritis.

ARF6-dependent endocytic trafficking of the Interferon-γ receptor drives adaptive immune resistance in cancer.

Adaptive immune resistance (AIR) is a protective process used by cancer to escape elimination by CD8+ T cells. Inhibition of immune checkpoints PD-1 and CTLA-4 specifically target Interferon-gamma (IFNγ)-driven AIR. AIR begins at the plasma membrane where tumor cell-intrinsic cytokine signaling is initiated. Thus, plasma membrane remodeling by endomembrane trafficking could regulate AIR. Herein we report that the trafficking protein ADP-Ribosylation Factor 6 (ARF6) is critical for IFNγ-driven AIR. ARF6 prevents transport of the receptor to the lysosome, augmenting IFNγR expression, tumor intrinsic IFNγ signaling and downstream expression of immunosuppressive genes. In murine melanoma, loss of ARF6 causes resistance to immune checkpoint blockade (ICB). Likewise, low expression of ARF6 in patient tumors correlates with inferior outcomes with ICB. Our data provide new mechanistic insights into tumor immune escape, defined by ARF6-dependent AIR, and support that ARF6-dependent endomembrane trafficking of the IFNγ receptor influences outcomes of ICB.

Maternal infection causes dysfunctional BCR signaling in male offspring due to aberrant Xist expression.

Infections during pregnancy with pathogens such as helminths correlate with altered immune responses to common childhood immunizations. However, the molecular mechanisms that underlie this remain unknown. Using our murine model of maternal schistosomiasis, when immunized, males from infected mothers had a lower frequency of antigen-specific germinal center B cells and downregulation of transcripts downstream of BCR signaling compared to males from uninfected mothers. This is driven by a reduction in developing B cell populations within the bone marrow of pups from infected mothers. Males from infected mothers were impacted to a greater extent than their female littermate counterparts. We found this defect to be caused by aberrant expression of the long non-coding RNA Xist in males leading to dysregulated Igα expression on developing B cells. This, for the first time, links dysfunctional BCR signaling with Xist expression, while also proposing a detrimental function for Xist expression in males.

Altered Offspring Immunity in Maternal Parasitic Infections.

Maternal infection during pregnancy is known to alter the development and function of offspring's immune system, leading to inappropriate immune responses to common childhood infections and immunizations. Although this is an expanding field, maternal parasitic infections remain understudied. Millions of women of reproductive age are currently at risk for parasitic infection, whereas many pregnant, chronically infected women are excluded from mass drug administration due partially to a lack of resources, as well as fear of unknown adverse fetal developmental outcomes. In areas endemic for multiple parasitic infections, such as sub-Saharan Africa, there are increased rates of morbidity and mortality for various infections during early childhood in comparison with nonendemic areas. Despite evidence supporting similar immunomodulatory effects between various parasite species, there is no clear mechanistic understanding of how maternal infection reprograms offspring immunity. This brief review will compare the effects of selected maternal parasitic infections on offspring immunity.

A subset of follicular helper-like MAIT cells can provide B cell help and support antibody production in the mucosa.

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that aid in protection against bacterial pathogens at mucosal surfaces through the release of inflammatory cytokines and cytotoxic molecules. Recent evidence suggests that MAIT cells can also provide B cell help. In this study, we describe a population of CXCR5+ T follicular helper (Tfh)­like MAIT cells (MAITfh) that have the capacity to provide B cell help within mucosal lymphoid organs. MAITfh cells are preferentially located near germinal centers in human tonsils and express the classical Tfh-associated transcription factor, B cell lymphoma 6 (BCL-6), the costimulatory markers inducible T cell costimulatory (ICOS) and programmed death receptor 1 (PD-1), and interleukin-21 (IL-21). We demonstrate the ability of MAIT cells to provide B cell help in vivo after mucosal challenge with Vibrio cholerae. Specifically, we show that adoptive transfer of MAIT cells into αß T cell­deficient mice promoted B cell differentiation and increased serum V. cholerae­specific IgA responses. Our data demonstrate the capacity of MAIT cells to participate in adaptive immune responses and suggest that MAIT cells may be potential targets for mucosal vaccines.

Maternal schistosomiasis impairs offspring Interleukin-4 production and B cell expansion.

Epidemiological studies have identified a correlation between maternal helminth infections and reduced immunity to some early childhood vaccinations, but the cellular basis for this is poorly understood. Here, we investigated the effects of maternal Schistosoma mansoni infection on steady-state offspring immunity, as well as immunity induced by a commercial tetanus/diphtheria vaccine using a dual IL-4 reporter mouse model of maternal schistosomiasis. We demonstrate that offspring born to S. mansoni infected mothers have reduced circulating plasma cells and peripheral lymph node follicular dendritic cells at steady state. These reductions correlate with reduced production of IL-4 by iNKT cells, the cellular source of IL-4 in the peripheral lymph node during early life. These defects in follicular dendritic cells and IL-4 production were maintained long-term with reduced secretion of IL-4 in the germinal center and reduced generation of TFH, memory B, and memory T cells in response to immunization with tetanus/diphtheria. Using single-cell RNASeq following tetanus/diphtheria immunization of offspring, we identified a defect in cell-cycle and cell-proliferation pathways in addition to a reduction in Ebf-1, a key B-cell transcription factor, in the majority of follicular B cells. These reductions are dependent on the presence of egg antigens in the mother, as offspring born to single-sex infected mothers do not have these transcriptional defects. These data indicate that maternal schistosomiasis leads to long-term defects in antigen-induced cellular immunity, and for the first time provide key mechanistic insight into the factors regulating reduced immunity in offspring born to S. mansoni infected mothers.

Metabolic reprogramming of the myeloid lineage by Schistosoma mansoni infection persists independently of antigen exposure.

Macrophages have a defined role in the pathogenesis of metabolic disease and cholesterol metabolism where alternative activation of macrophages is thought to be beneficial to both glucose and cholesterol metabolism during high fat diet induced disease. It is well established that helminth infection protects from metabolic disease, but the mechanisms underlying protection are not well understood. Here, we investigated the effects of Schistosoma mansoni infection and cytokine activation in the metabolic signatures of bone marrow derived macrophages using an approach that integrated transcriptomics, metabolomics, and lipidomics in a metabolic disease prone mouse model. We demonstrate that bone marrow derived macrophages (BMDM) from S. mansoni infected male ApoE-/- mice have dramatically increased mitochondrial respiration compared to those from uninfected mice. This change is associated with increased glucose and palmitate shuttling into TCA cycle intermediates, increased accumulation of free fatty acids, and decreased accumulation of cellular cholesterol esters, tri and diglycerides, and is dependent on mgll activity. Systemic injection of IL-4 complexes is unable to recapitulate either reductions in systemic glucose AUC or the re-programing of BMDM mitochondrial respiration seen in infected males. Importantly, the metabolic reprogramming of male myeloid cells is transferrable via bone marrow transplantation to an uninfected host, indicating maintenance of reprogramming in the absence of sustained antigen exposure. Finally, schistosome induced metabolic and bone marrow modulation is sex-dependent, with infection protecting male, but not female mice from glucose intolerance and obesity. Our findings identify a transferable, long-lasting sex-dependent reprograming of the metabolic signature of macrophages by helminth infection, providing key mechanistic insight into the factors regulating the beneficial roles of helminth infection in metabolic disease.

IL-4 promotes stromal cell expansion and is critical for development of a type-2, but not a type 1 immune response.

IL-4 is critical for differentiation of Th2 cells and antibody isotype switching, but our work demonstrated that it is produced in the peripheral LN under both Type 2, and Type 1 conditions, raising the possibility of other functions. We found that IL-4 is vital for proper positioning of hematopoietic and stromal cells in steady state, and the lack of IL-4 or IL-4Rα correlates with disarrangement of both follicular dendritic cells and CD31+ endothelial cells. We observed a marked disorganization of B cells in these mice, suggesting that the lymphocyte-stromal cell axis is maintained by the IL-4 signaling pathway. This study showed that absence of IL-4 correlates with significant downregulation of Lymphotoxin alpha (LTα) and Lymphotoxin beta (LTß), critical lymphokines for the development and maintenance of lymphoid organs. Moreover, immunization of IL-4 deficient mice with Type 2 antigens failed to induce lymphotoxin production, LN reorganization, or germinal center formation, while this process is IL-4 independent following Type 1 immunization. Additionally, we found that Type 1 antigen mediated LN reorganization is dependent on IFN-γ in the absence of IL-4. Our findings reveal a role of IL-4 in the maintenance of peripheral lymphoid organ microenvironments during homeostasis and antigenic challenge.

Schistosoma mansoni Infection-Induced Transcriptional Changes in Hepatic Macrophage Metabolism Correlate With an Athero-Protective Phenotype.

Hepatic macrophages play an essential role in the granulomatous response to infection with the parasitic helminth Schistosoma mansoni, but the transcriptional changes that underlie this effect are poorly understood. To explore this, we sorted the two previously recognized hepatic macrophage populations (perivascular and Kupffer cells) from naïve and S. mansoni-infected male mice and performed microarray analysis as part of the Immunological Genome Project. The two hepatic macrophage populations exhibited remarkably different genomic profiles. However, this diversity was substantially reduced following infection with S. mansoni, and in fact, both populations demonstrated increases in transcripts of the monocyte lineage, suggesting that both populations may be replenished by monocytes following infection. Pathway analysis showed a profound alteration in global metabolic pathways, including changes to phospholipid and cholesterol metabolism, as well as amino acid biosynthesis and glucagon signaling. These changes suggest a possible mechanism for the previously reported athero-protective effects of S. mansoni infection. Indeed, we find that male ApoE null mice fed a high-fat diet in combination with S. mansoni infection have reduced plaque area and increased glucose tolerance as compared to control mice. Transcript analysis of infected and control high-fat diet fed ApoE-/- mice confirm that ApoC1, Psat1, and Gys1 are all altered by infection, suggesting that altered hepatic macrophage metabolism is associated with S. mansoni- induced protection from hyperlipidemia, atherosclerosis, and glucose intolerance. These results suggest a previously unknown and unreported role of hepatic macrophages in the modulation of whole body lipid and glucose metabolism during infection and provide a template for examining the role of immunomodulation on the long-term metabolism of the host.

Vitamin D Receptor-Dependent Signaling Protects Mice From Dextran Sulfate Sodium-Induced Colitis.

Low vitamin D status potentiates experimental colitis, but the vitamin D-responsive cell in colitis has not been defined. We hypothesized that vitamin D has distinct roles in colonic epithelial cells and in nonepithelial cells during colitis. We tested this hypothesis by using mice with vitamin D receptor (VDR) deletion from colon epithelial cells (CEC-VDRKO) or nonintestinal epithelial cells (NEC-VDRKO). Eight-week-old mice were treated with 1.35% dextran sulfate sodium (DSS) for 5 days and then euthanized 2 or 10 days after removal of DSS. DSS induced body weight loss and increased disease activity index and spleen size. This response was increased in NEC-VDRKO mice but not CEC-VDRKO mice. DSS-induced colon epithelial damage and immune cell infiltration scores were increased in both mouse models. Although the epithelium healed between 2 and 10 days after DSS administration in control and CEC-VDRKO mice, epithelial damage remained high in NEC-VDRKO mice 10 days after removal of DSS, indicating delayed epithelial healing. Gene expression levels for the proinflammatory, M1 macrophage (Mɸ) cytokines tumor necrosis factor-α, nitric oxide synthase 2, and interleukin-1ß were significantly elevated in the colon of NEC-VDRKO mice at day 10. In vitro experiments in murine peritoneal Mɸs demonstrated that 1,25 dihydroxyvitamin D directly inhibited M1 polarization, facilitated M2 polarization, and regulated Mɸ phenotype switching toward the M2 and away from the M1 phenotype. Our data revealed unique protective roles for vitamin D signaling during colitis in the colon epithelium as well as nonepithelial cells in the colon microenvironment (i.e., modulation of Mɸ biology).

Migratory CD103+ dendritic cells suppress helminth-driven type 2 immunity through constitutive expression of IL-12.

CD8α(+) and CD103(+) dendritic cells (DCs) play a central role in the development of type 1 immune responses. However, their role in type 2 immunity remains unclear. We examined this issue using Batf3(-/-) mice, in which both of these DC subsets are missing. We found that Th2 cell responses, and related events such as eosinophilia, alternative macrophage activation, and immunoglobulin class switching to IgG1, were enhanced in Batf3(-/-) mice responding to helminth parasites. This had beneficial or detrimental consequences depending on the context. For example, Batf3 deficiency converted a normally chronic intestinal infection with Heligmosomoides polygyrus into an infection that was rapidly controlled. However, liver fibrosis, an IL-13-mediated pathological consequence of wound healing in chronic schistosomiasis, was exacerbated in Batf3(-/-) mice infected with Schistosoma mansoni. Mechanistically, steady-state production of IL-12 by migratory CD103(+) DCs, independent of signals from commensals or TLR-initiated events, was necessary and sufficient to exert the suppressive effects on Th2 response development. These findings identify a previously unrecognized role for migratory CD103(+) DCs in antagonizing type 2 immune responses.

IL-4-secreting secondary T follicular helper (Tfh) cells arise from memory T cells, not persisting Tfh cells, through a B cell-dependent mechanism.

Humoral immunity requires cross-talk between T follicular helper (Tfh) cells and B cells. Nevertheless, a detailed understanding of this intercellular interaction during secondary immune responses is lacking. We examined this by focusing on the response to a soluble, unadjuvanted, pathogen-derived Ag (soluble extract of Schistosoma mansoni egg [SEA]) that induces type 2 immunity. We found that activated Tfh cells persisted for long periods within germinal centers following primary immunization. However, the magnitude of the secondary response did not appear to depend on pre-existing Tfh cells. Instead, Tfh cell populations expanded through a process that was dependent on memory T cells recruited into the reactive LN, as well as the participation of B cells. We found that, during the secondary response, IL-4 was critical for the expansion of a population of plasmablasts that correlated with increased SEA-specific IgG1 titers. Additionally, following immunization with SEA (but not with an Ag that induced type 1 immunity), IL-4 and IL-21 were coproduced by individual Tfh cells, revealing a potential mechanism through which appropriate class-switching can be coupled to plasmablast proliferation to enforce type 2 immunity. Our findings demonstrate a pivotal role for IL-4 in the interplay between T and B cells during a secondary Th2 response and have significant implications for vaccine design.

Molecular cloning and characterization of a nematode polyprotein antigen/allergen from the human and animal hookworm Ancylostoma ceylanicum.

Nematodes are unable to synthesize fatty acids de novo and must acquire them from the environment or host. It is hypothesized that two unique classes of fatty acid and retinol binding proteins that nematodes produce (fatty acid and retinol binding (FAR) and nematode polyprotein antigen/allergen (NPA)) are used to meet this need. A partial cDNA has been cloned corresponding to four subunits of a putative Ancylostoma ceylanicum NPA (AceNPA). The translated amino acid sequence of AceNPA shares sequence identity with similar proteins from Dictyocaulus viviparus, Ascaris suum, and Ostertagia ostertagi. Immunoblot experiments using a polyclonal anti-AceNPA IgG revealed proteins corresponding to the expected sizes of single, as well as two or three un-cleaved NPA subunits in adult excretory/secretory proteins and soluble adult worm extracts. Immunohistochemistry experiments localize AceNPA to the cuticle, pseudocoelomic space and testes suggesting a role in hookworm biology that is distinct from what has previously been defined for other hookworm lipid binding proteins. A single recombinant subunit of AceNPA (rAceNPAb) demonstrated binding in vitro to fluorescent fatty acids DAUDA, cis-parinaric acid, as well as retinol, at equilibrium dissociation constants in the low micromolar range. Further, in vitro data reveal that rAceNPAb binds fatty acids with chain lengths of C12-C22, with the greatest affinities for arachidonic, linoleic (C18), and eicosapentaenoic (C20) acids.

Regulation of the development of the hepatic B cell compartment during Schistosoma mansoni infection.

During infection with the helminth parasite Schistosoma mansoni, Ab regulates hepatic inflammation, and local production of Ig in the liver appears to play a role in this process. Exploring the development of the B cell response during infection, we found that parasite-specific IgG1-secreting plasma cells appeared first in the hepatic and mesenteric lymph nodes (LNs) and then at later times in the spleen, liver, and bone marrow. The LN B cell population peaked between weeks 10 and 12 of infection, and then contracted at a time that coincided with the expansion of the hepatic IgG1(+) B cell compartment, suggesting that B cells migrate from LNs to liver. CXCL9 and -16 expression in the liver increased during the time frame of B cell recruitment. Expression of the CXCL16 receptor CXCR6 was increased on B cells within the hepatic LNs, but not the mesenteric LNs. CXCR3, the receptor for CXCL9, was broadly expressed on IgG1(+) B cells in LNs and liver during infection. Increased hepatic expression of CXCL9 and -16 failed to occur if the IL-10R was blocked in vivo, an intervention associated with decreased liver B cell infiltration and the development of severe disease. Hepatic LN IgG1(+) cells migrated toward CXCL9 and -16 in vitro and to the liver in a pertussis toxin-sensitive fashion. Our data suggest that the coordinated expression of CXCL9 and -16 in the liver and of CXCR6 and CXCR3 on responding B cells within the hepatic LNs underpins establishment of the hepatic B cell infiltrate during chronic schistosomiasis.

IL-10R blockade during chronic schistosomiasis mansoni results in the loss of B cells from the liver and the development of severe pulmonary disease.

In schistosomiasis patients, parasite eggs trapped in hepatic sinusoids become foci for CD4+ T cell-orchestrated granulomatous cellular infiltrates. Since the immune response is unable to clear the infection, the liver is subjected to ongoing cycles of focal inflammation and healing that lead to vascular obstruction and tissue fibrosis. This is mitigated by regulatory mechanisms that develop over time and which minimize the inflammatory response to newly deposited eggs. Exploring changes in the hepatic inflammatory infiltrate over time in infected mice, we found an accumulation of schistosome egg antigen-specific IgG1-secreting plasma cells during chronic infection. This population was significantly diminished by blockade of the receptor for IL-10, a cytokine implicated in plasma cell development. Strikingly, IL-10R blockade precipitated the development of portal hypertension and the accumulation of parasite eggs in the lungs and heart. This did not reflect more aggressive Th2 cell responsiveness, increased hepatic fibrosis, or the emergence of Th1 or Th17 responses. Rather, a role for antibody in the prevention of severe disease was suggested by the finding that pulmonary involvement was also apparent in mice unable to secrete class switched antibody. A major effect of anti-IL-10R treatment was the loss of a myeloid population that stained positively for surface IgG1, and which exhibited characteristics of regulatory/anti-inflammatory macrophages. This finding suggests that antibody may promote protective effects within the liver through local interactions with macrophages. In summary, our data describe a role for IL-10-dependent B cell responses in the regulation of tissue damage during a chronic helminth infection.

Characterisation of a fatty acid and retinol binding protein orthologue from the hookworm Ancylostoma ceylanicum.

Hookworms, bloodfeeding intestinal nematodes, infect nearly one billion people in resource limited countries and are a leading cause of anaemia and malnutrition. Like other nematodes, hookworms lack the capacity to synthesise essential fatty acids de novo and therefore must acquire those from exogenous sources. The cDNA corresponding to a putative Ancylostoma ceylanicum fatty acid and retinol binding protein-1 (AceFAR-1) was amplified from adult hookworm mRNA. Studies using quantitative reverse transcriptase real-time PCR demonstrate that AceFAR-1 transcripts are most abundant in the earliest developmental stages of the parasite, and greater in females than males. Using in vitro assays, the recombinant AceFAR-1 (rAceFAR-1) was shown to bind individual fatty acids with equilibrium dissociation constants in the low micromolar range. The pattern of fatty acid uptake by live adult worms cultured ex vivo was similar to the in vitro binding profile of rAceFAR-1, raising the possibility that the native protein may be involved in acquisition of fatty acids by A. ceylanicum. Animals vaccinated orally with rAceFAR-1 and the mucosal adjuvant cholera toxin exhibited a statistically significant (40-47%) reduction in intestinal worm burden compared with controls immunized with antigen or adjuvant alone. Together, these data suggest a potential role for AceFAR-1 in hookworm biology, making it a potentially valuable target for drug and vaccine development.