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Interactions with Ag-specific T cells drive B cell activation and fate choices that ultimately determine the quality of high-affinity Ab responses. As such, these interactions, and especially the long-lived interactions that occur before germinal center formation, may be important checkpoints to regulate undesirable responses. Using mouse model Ag systems, we directly observed interactions between T and B cells responding to the self-antigen myelin oligodendrocyte glycoprotein (MOG) and found that they are of lower quality compared with interactions between cells responding to the model foreign Ag nitrophenyl-haptenated OVA. This was associated with reduced expression of molecules that facilitate these interactions on the B cells, but not on T cells. B cell expression of these molecules was not dictated by the T cell partner, nor could the relative lack of expression on MOG-specific (MOG-sp.) B cells be reversed by a multivalent Ag. Instead, MOG-sp. B cells were inherently less responsive to BCR stimulation than MOG-non-sp. cells. However, the phenotype of MOG-sp. B cells was not consistent with previous descriptions of autoimmune B cells that had been tolerized via regular exposure to systemically expressed self-antigen. This suggests that alternate anergy pathways may exist to limit B cell responses to tissue-restricted self-antigens.

Staphylococcus aureus is a foremost bacterial pathogen responsible for a vast array of human diseases. Staphylococcal superantigens (SAgs) constitute a family of potent exotoxins secreted by S. aureus, and SAg genes are found ubiquitously in human isolates. SAgs bind directly to MHC class II molecules and T cell receptors, driving extensive T cell activation and cytokine release. Although these toxins have been implicated in serious disease including toxic shock syndrome, we aimed to further elucidate the mechanisms by which SAgs contribute to staphylococcal pathogenesis during septic bloodstream infections. As most conventional mouse strains respond poorly to staphylococcal SAgs, we utilized transgenic mice encoding humanized MHC class II molecules (HLA-DR4) as these animals are much more susceptible to SAg activity. Herein, we demonstrate that SAgs contribute to the severity of S. aureus bacteremia by increasing bacterial burden, most notably in the liver. We established that S. aureus bloodstream infection severity is mediated by CD4+ T cells and interferon-gamma (IFNγ) is produced to very high levels during infection in a SAg-dependent manner. Bacterial burden and disease severity were reduced by antibody blocking of IFNγ, phenocopying isogenic SAg deletion mutant strains. Additionally, cytokine analysis demonstrated that the immune system was skewed towards a proinflammatory response that was reduced by IFNγ blocking. Infection kinetics and flow cytometry analyses suggested this was a macrophage driven mechanism, which was confirmed through macrophage depletion experiments. Further validation with human leukocytes indicated that excessive IFNγ allowed S. aureus to replicate at a higher rate within macrophages. Together, this suggests that SAgs promote S. aureus survival by manipulating immune responses that would otherwise be effective at clearing S. aureus. This work implicates SAg toxins as critical targets for preventing persistent or severe S. aureus disease.

Intracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. In this study, we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in retinoic acid–related orphan receptor γt+ cells and conventional dendritic cells (DCs) using germline and conditional double knockout mice. Although Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells in the intestinal mucosa, we found that they were required in conventional DCs for optimal Ag processing and presentation to CD4+ T cells. Using a real-time imaging method, we show that TMEM176A/B accumulate in dynamic post-Golgi vesicles preferentially linked to the late endolysosomal system and strongly colocalize with HLA-DM. Taken together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC class II compartment of DCs for the fine regulation of Ag presentation and naive CD4+ T cell priming.

Throughout the developing GC response, B cell survival and fate choices made at the single cell level are dependent on signals received largely through interactions with other cells, often with cognate T cells. The type of signals that a given B cell can encounter is dictated by its location within tissue microarchitecture. The focus of this review is on the initiation and evolution of the GC response at the earliest time points. Here, we review the key factors influencing the progression of GC B cell differentiation that are both stage and context dependent. Finally, we describe the coevolution of niches within and surrounding the GC that influence the outcome of the GC response.

Background Patients with MS have an altered gut microbiota compared to healthy individuals, as well as elevated small intestinal permeability, which may be contributing to the development and progression of the disease. Objective We sought to investigate if fecal microbiota transplantation was safe and tolerable in MS patients and if it could improve abnormal intestinal permeability. Methods Nine patients with MS were recruited and provided monthly FMTs for up to six months. The primary outcome investigated was change in peripheral blood cytokine concentrations. The secondary outcomes were gut microbiota composition, intestinal permeability, and safety (assessed with EDSS and MRI). Results The study was terminated early and was subsequently underpowered to assess whether peripheral blood cytokines were altered following FMTs. FMTs were safe in this group of patients. Two of five patients had elevated small intestinal permeability at baseline that improved to normal values following FMTs. Significant, donor-specific, beneficial alterations to the MS patient gut microbiota were observed following FMT. Conclusion FMT was safe and tolerable in this cohort of RRMS patients, may improve elevated small intestinal permeability, and has the potential to enrich for an MS-protective microbiota. Further studies with longer follow-up and larger sample sizes are required to determine if FMT is a suitable therapy for MS.

Anti-CD20 B cell depleting therapies have demonstrated that B cells are important drivers of disease progress in Multiple Sclerosis, although the pathogenic mechanisms are not well understood. A population of B cells accumulates in the inflamed meninges in MS and also some chronic animal models of disease, typically adjacent to demyelinating lesions. The role of these meningeal B cells in disease is not known, nor is their susceptibility to anti-CD20 therapy. Here, we administered anti-CD20 to 2D2 IgHMOG spontaneous experimental autoimmune encephalomyelitis mice in the chronic phase of disease, after the establishment of meningeal B cell clusters. Compared to the circulation, lymph nodes, and spleen, B cell depletion from the CNS was delayed and not evident until 7d post administration of anti-CD20. Further, we did not find evidence that anti-CD20 accessed meningeal B cells directly, but rather that depletion was indirect and the result of ongoing turnover of the meningeal population and elimination of the peripheral pool from which it is sustained. The reduction of B cell numbers in the CNS coincided with less demyelination of the spinal cord white matter and also, surprisingly, an increase in the number of T cells recruited to the meninges but not parenchyma.

SummaryIntracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. Here we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in RORγt+ cells and conventional dendritic cells (cDCs) using germline and conditional double knock-out (DKO) mice. While Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells (ILC3s) in the intestinal mucosa, we found that they were required in cDCs for optimal antigen processing and presentation to CD4+ T cells. Using a real-time imaging method, we show that TMEM176A/B accumulate in dynamic post-Golgi vesicles preferentially linked to the late endolysosomal system and strongly colocalize with HLA-DM. Together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC II compartment (MIIC) of DCs for the fine regulation of antigen presentation and naive CD4+ T cell priming.

Background Colorectal cancer (CRC) is the second leading cause of cancer death, with a major risk factor being chronic inflammation. Despite the clear association between inflammation and cancer, the mechanism by which colitis leads to CRC is not well understood. We recently showed that the presence of inflammation does not always correlate with colonic tumorigenesis, as the type of colitis (i.e. colitis-inducing agent) appears to be important for tumor initiation. Aims In this study, we aim to explore the mechanism by which inflammation contributes to the initiation of colitis-associated cancer. We hypothesized that dextran sodium sulfate (DSS)-induced colitis leads to the infiltration of a specific immune cell type that is associated with colonic tumorigenesis. Methods To generate tamoxifen-inducible Cre transgenic mice that allow for Dclk1+ cell lineage tracing and cell-specific knock-out of the tumor suppressor adenomatous polyposis coli (APC), we first crossed our transgenic Dclk1CreERT2 mice to both ROSA26tdTomato and APCfl/fl mice (Dclk1/APCfl/fl). Following Tamoxifen induction, mice were treated with the colitis-inducing agents DSS, trinitrobenzene sulfonic acid (TNBS), oxazolone, or Citrobacter rodentium. The tumor studies were repeated using azoxymethane (AOM)-DSS induced colitis-associated cancer model. Results Treatment with any of the four colitis-inducing agents (DSS, TNBS, oxazolone, or C. rodentium) induced colonic inflammation as detected by increased myeloperoxidase (MPO) activity and histologic analysis. Interestingly, DSS administration led to colonic tumors, whereas TNBS, oxazolone, or C. rodentium did not, even up to 52 weeks following colitis induction. FACS analysis of immune cells in the colon revealed no difference in the number of T or B cells in mice treated with any of the colitis-inducing agents. We did, however, detect significantly increased levels of Ly6G+ neutrophils and F4/80+ macrophages in DSS-treated mice compared to mice in any of the other three models of colitis. Consistent with this myeloid cell infiltration, significantly upregulated protein levels of G-CSF, IL-6, TNF-α, and CXCL1 were detected in DSS-treated mice compared to the other three models of colitis. IL-1α, IL-1β, IL-4, IL-10, and TGF-β levels were unchanged. Conclusions Our data suggest that infiltration of Ly6G+ neutrophils and pro-inflammatory F4/80+ macrophages, unique to DSS-induced colitis, contributes to colonic tumor formation. These data demonstrate that specific immune cell types, rather than the presence of colonic inflammation, play a critical role in the initiation of colitis-associated CRC. Funding Agencies CAG, CIHR

We examined the unique contributions of the cytokines IL-21 and IL-4 on germinal center (GC) B cell initiation and subsequent maturation in a murine model system. Similar to other reports, we found T follicular helper cell expression of IL-21 begins prior to T follicular helper cell migration into the B cell follicle and precedes that of IL-4. Consistent with this timing, IL-21 signaling has a greater influence on the perifollicular pre-GC B cell transition to the intrafollicular stage. Notably, Bcl6hi B cells can form in the combined absence of IL-21R– and STAT6-derived signals; however, these nascent GC B cells cease to proliferate and are more prone to apoptosis. When B cells lack either IL-21R or STAT6, aberrant GCs form atypical centroblasts and centrocytes that differ in their phenotypic maturation and costimulatory molecule expression. Thus, IL-4 and IL-21 play nonredundant roles in the phased progression of GC B cell development that can initiate in the combined absence of these cytokine signals.

Background Colorectal cancer (CRC) is the third leading cause of cancer death, with a major risk factor being chronic inflammation. Thus, patients with inflammatory bowel disease (IBD) are at an increased risk of CRC. Despite the clear association between inflammation and cancer, the mechanism by which colitis leads to CRC is still not well understood. Aims In this study, we aim to explore the mechanism by which inflammation contributes to the initiation of colitis-associated cancer (CAC). We hypothesize that dextran sodium sulfate (DSS)-induced colitis leads to the infiltration of a specific immune cell type associated with tumorigenesis. Methods Following an injection of azoxymethane (AOM), mice were treated with the colitis-inducing agents DSS, trinitrobenzene sulfonic acid (TNBS), oxazolone (oxa), Citrobacter rodentium, or Doxorubicin (Doxo). The tumor studies were repeated using our published Cre-dependent murine model of CAC. To generate tamoxifen-inducible Cre transgenic mice that allow for Dclk1+ cell lineage tracing and cell-specific knock-out of the tumor suppressor adenomatous polyposis coli (APC), we crossed our Dclk1CreERT2 mice to both ROSA26tdTomato and APCfl/fl mice (Dclk1/APCfl/fl). Results Treatment with DSS, TNBS, oxa, C. rodentium, or Doxo induced colonic inflammation as detected by increased myeloperoxidase (MPO) activity and histologic analysis. DSS administration led to colonic tumors, whereas TNBS, oxa, C. rodentium, or Doxo did not lead to tumorigenesis up to 52 weeks following colitis induction. Upon flow cytometric analysis of several types of immune cells in the colonic tissue, we observed no difference in the number of T and B cells between mice treated with various colitis inducing agents. We did, however, detect significantly increased levels of Ly6G+ neutrophils and F4/80+Ly6Chi macrophages in the DSS-treated mice when compared to mice in the other models of colitis. mRNA and protein array analyses of the colonic tissue, as well as analysis of the RNA-seq data from 206 UC patients (GSE109142), revealed upregulated expression of genes associated with macrophages and neutrophils. Addition of macrophage-produced cytokines, such as IL-1β, TNF-α, or IL-6, induced lineage tracing of Dclk1+ tuft cells in intestinal organoids. Clodronate liposome-mediated depletion of F4/80+Ly6Chi macrophages significantly reduced the number of colonic tumors but did not affect tumor size in Dclk1/APCfl/fl mice. Conclusions Our data suggest that infiltration of F4/80+Ly6Chi macrophages, unique to DSS-induced colitis, leads to colonic tumor formation. This demonstrates that specific immune cell types, rather than the presence of colonic inflammation, plays an important role in the initiation of CAC. Funding Agencies CAG, CIHR

Once activated, T cells gain the ability to access both healthy and inflamed nonlymphoid tissues. They are then reactivated to remain in the tissue and exert their effector function only if they encounter their specific Ag. In this study, we set out to determine if the same is true for B cells using a mouse model of CNS autoimmunity that incorporates both T and B cell recognition of a myelin autoantigen. Both T and B cells were common infiltrates of spinal cords in diseased mice. However, unlike T cells, anti-myelin B cells were excluded from the inflamed tissue. Further, CNS B cells did not have a phenotype consistent with Ag-specific activation as it occurs in lymphatic tissue. Instead, they expressed elevated levels of CD80, indicating that B cells may contribute to local inflammation through nonantigen-specific mechanisms.

Pathogenic lymphocytes aberrantly recognize and mount an immune response against self-antigens, leading to the destruction of healthy cells, tissues and organs. Recent studies have shown that both B and T lymphocytes contribute to the development, prevention and modulation of various autoimmune diseases. Regulatory T and B cell subsets appear to play a prominent role in the prevention of autoimmune diseases. The recent identification of novel regulatory Th17 cells, termed as Treg17 cells, has expanded the scope of regulatory T lymphocytes (Treg cells) in the prevention of autoimmune diseases. Similarly, novel regulatory B cell subsets, termed as Breg cells, acting on their own or by inducing Treg cells have extended the role of B lymphocytes in the prevention and regulation of autoimmune diseases. We suggest that Treg17 cells and Breg cells have an important immunoregulatory role in autoimmune diseases.

Summary: B cell fate decisions within a germinal center (GC) are critical to determining the outcome of the immune response to a given antigen. Here, we characterize GC kinetics and B cell fate choices in a response to the autoantigen myelin oligodendrocyte glycoprotein (MOG) and compare the response with a standard model foreign antigen. Both antigens generate productive primary responses, as evidenced by GC development, circulating antigen-specific antibodies, and differentiation of memory B cells. However, in the MOG response, the status of the cognate T cell partner drives preferential B cell differentiation to a memory phenotype at the expense of GC maintenance, resulting in a truncated GC. Reduced plasma cell differentiation is largely independent of T cell influence. Interestingly, memory-phenotype B cells formed in the MOG GC are not long lived, resulting in a failure of the B cell response to secondary challenge. : Jain et al. compare B cell responses to different antigens and find that they preferentially differentiate into short-lived memory-phenotype B cells in response to a myelin autoantigen. This was in part controlled by T cells and influenced by TCR affinity for antigen, demonstrating that antigen characteristics can determine immune outcome. Keywords: B cell, T follicular helper cell, autoimmunity, germinal center, MOG, memory B cell, plasma cell, multiple sclerosis

B cell fate decisions within a germinal center (GC) are critical to determining the outcome of the immune response to a given antigen. Here, we characterize GC kinetics and B cell fate choices in a response to the autoantigen myelin oligodendrocyte glycoprotein (MOG), and compare them the response to a standard model foreign antigen (NP-haptenated ovalbumin, NPOVA). Both antigens generated productive primary responses, as evidenced by GC development, circulating antigen-specific antibodies, and differentiation of memory B cells. However, in the MOG response the status of the cognate T cell partner drove preferential B cell differentiation to a memory phenotype at the expense of GC maintenance, resulting in a truncated GC. Reduced plasma cell differentiation was largely independent of T cell influence. Interestingly, memory B cells formed in the MOG GC were unresponsive to secondary challenge and this could not be overcome with T cell help.

We propose that there is a special B-1a B cell subset ("sB-1a" cells) that mediates linked processes very early after immunization to initiate cutaneous contact sensitivity (CS), delayed-type hypersensitivity (DTH), and immune resistance to pneumococcal pneumonia. Our published data indicate that in CS and DTH, these initiating processes are required for elicitation of the delayed onset and late-occurring classical T cell-mediated responses. sB-1a cells resemble memory B2 cells, as they are stimulated within 1 h of immunization and depend on T helper cytokines-uniquely IL-4 from hepatic iNKT cells--for activation and rapid migration from the peritoneal cavity to the spleen to secrete IgM antibody (Ab) and Ab-derived free light chains (FLCs) by only 1 day after immunization. Unlike conventional B-1a (cB-1a) cell-produced IgM natural Ab, IgM Ab produced by sB-1a cells has high Ag affinity owing to immunoglobulin V-region mutations induced by activation-induced cytidine deaminase (AID). The dominant cB-1a cells are increased in immunized AID-deficient mice but do not mediate initiation, CS, or pneumonia resistance because natural Ab has relatively low Ag affinity because of unmutated germ-line V regions. In CS and DTH, sB-1a IgM Ag affinity is sufficiently high to mediate complement activation for generation of C5a that, together with vasoactive mediators such as TNF-α released by FLC-sensitized mast cells, activate local endothelium for extravascular recruitment of effector T cells. We conclude by discussing the possibility of functional sB-1 cells in humans.

Neutrophils contribute to demyelinating autoimmune diseases, yet their phenotype and functions have been elusive to date. Here, we demonstrate that ICAM1 surface expression distinguishes extra- from intravascular neutrophils in the mouse CNS during experimental autoimmune encephalomyelitis (EAE). Transcriptomic analysis of these 2 subpopulations indicated that neutrophils, once extravasated, acquire macrophage-like properties, including the potential for immunostimulation and MHC class II-mediated antigen presentation. In corroboration, super-resolution (3D stimulated emission-depletion [STED]) microscopy revealed neutrophils forming synapses with T and B cells in situ. Further, neutrophils specifically express the aspartic retroviral-like protease ASPRV1, which increases in the CNS during EAE and severe cases of multiple sclerosis. Without ASPRV1, mice immunized with a new B cell-dependent myelin antigen (but not with the traditional myelin oligodendrocyte glycoprotein peptide) develop a chronic phase of EAE that is less severe and even completely fades in many individuals. Therefore, ICAM1+ macrophage-like neutrophils can play both shared and nonredundant roles in autoimmune demyelination, among them perpetuating inflammation via ASPRV1. CA extern

To reconcile conflicting reports on the role of CD40 signaling in germinal center (GC) formation, we examined the earliest stages of murine GC B cell differentiation. Peri-follicular GC precursors first expressed intermediate levels of BCL6 while co-expressing the transcription factors RelB and IRF4, the latter known to repress Bcl6 transcription. Transition of GC precursors to the BCL6hi follicular state was associated with cell division, although the number of required cell divisions was immunogen dose dependent. Potentiating T cell help or CD40 signaling in these GC precursors actively repressed GC B cell maturation and diverted their fate towards plasmablast differentiation, whereas depletion of CD4+ T cells promoted this initial transition. Thus while CD40 signaling in B cells is necessary to generate the immediate precursors of GC B cells, transition to the BCL6hi follicular state is promoted by a regional and transient diminution of T cell help. DOI: http://dx.doi.org/10.7554/eLife.19552.001

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

Prior studies of classical 24 h responses in TNP-Cl (picryl chloride) allergic contact sensitivity (CS), showed mediation by Th1 cells in CBA mice, and established that 24 h elicitation of responses requires an early 2 h CS-initiating component dependent on iNKT cells, IL-4 and B-1 B cells. Here, we studied the other form of cytotoxic T cell (Tc1) CS in DNFB sensitized BALB/c mice and determined that similar CS-initiation also is required. We systematically tested each step of the initiation pathway in this model. Thus, DNFB Tc1 CS was significantly impaired in iNKT cell deficient CD1d(-/-) and Jα18(-/-) mice, IL4Rα(-/-) and STAT-6(-/-) mice, and also in pan B-cell deficient JH(-/-) mice. Further, the Tc1 DNFB CS-initiating component, like Th1 response to TNP-Cl, was elicited by only 1-day after immunization, due to B-1 cells. In summary, we show that CS-Initiation also is required in Tc1 CS. Further, we have newly determined regulatory support of both the early and late components of DNFB induced Tc1 CS by iNKT cells and γδ-T cells. In summary, both iNKT cells and assisting γδ-T cells are involved in initiating and effector phases of DNFB induced CS.

Germinal centers (GC) are large aggregates of proliferating B lymphocytes within follicles of lymphoid tissue that form during adaptive immune responses. GCs are the source of long-lived B cells that form the basis for pathogen-specific lifelong B cell immunity. The complex architecture of these structures includes subdomains that differ significantly in their stromal cell and T lymphocyte subset composition. In part due to their structural complexity and potential to generate some lymphomas, much interest and many theories about GC dynamics have emerged. Here, we review recent research employing in vivo imaging that has begun to untangle some of the mysteries.

Contact sensitivity (CS) is related to delayed-type hypersensitivity and is a well-characterized prototype of T cell-mediated inflammation. However, the inflammatory response associated with CS is additionally dependent on Ag-specific IgM produced by a subpopulation of B cells in response to sensitization. Upon re-exposure to hapten, this IgM mediates rapid vascular activation and subsequent recruitment of proinflammatory T cells to the local site. Interference with this pathway prevents the full development of the classic delayed inflammatory response and is therefore termed the “CS initiation” pathway. In this study, we show that CS initiation is defective in mice deficient in activation-induced deaminase, an enzyme central to the process of somatic hypermutation. Using adoptive transfer experiments, we demonstrate that the defect is specific to a B-1-like population of B cells and that transfer of WT cells reconstitutes CS initiation mechanisms in deficient recipients. We went on to identify a novel subpopulation of Ag-binding B cells in the spleens of sensitized mice that possess initiation activity (CD19+CD5+Thy-1intIgMhighIgDhigh) that we name “initiator B cells.” Analysis of BCR H chain genes isolated from these cells revealed evidence of activation-induced deaminase-mediated somatic hypermutation. The sensitivity of CS initiation to very low amounts of sensitizing hapten suggests that the responsible B cells have increased IgM receptor gene mutations enabling selection to generate Abs with sufficient affinity to mediate the response.

Objective The capture of blood cells from the circulation is mediated by highly specialized adhesion molecules. These molecules contribute to the specificity of recruitment for various subsets. Here, we used a simple substrate of hyaluronic acid to investigate the specificity of CD44-mediated recruitment from human whole blood under shear conditions. Materials and Methods Human whole blood was perfused through a parallel-plate flow chamber, which mimics intravascular conditions. Microscopy was used to directly observe blood-cell interactions with adhesion molecule substrates. Results Erythrocytes, but not leukocytes, efficiently tethered to and rolled on the hyaluronic acid substrate. These interactions were demonstrated to be mediated by CD44 and regulated by the sialic acid content of the cells. Inflammatory stimuli did not result in enhanced erythrocyte rolling. Rather, interactions were restricted to aged erythrocytes approaching senescence. This mechanism of erythrocyte capture from the blood flow was found to be restricted to primates and not conserved across mammalian species. Conclusion This is the first report of erythrocyte tethering and rolling under shear conditions, a behavior, until now, thought to be exclusive to leukocytes. It may represent an important mechanism to identify, capture, and clear old erythrocytes during normal homeostasis or clot formation.

We characterized B cell infiltration of the spinal cord in a B cell-dependent, spontaneous model of central nervous system (CNS) autoimmunity that develops in a proportion of mice with mutant T and B cell receptors specific for myelin oligodendrocyte glycoprotein (MOG). We found that, while males are more likely to develop disease, females are more likely to have a chronic rather than monophasic disease course. B cell infiltration of the spinal cord was investigated by histology and FACs. CD4+ T cell infiltration was pervasive throughout the white and in some cases grey matter. B cells were almost exclusively restricted to the meninges, often in clusters reminiscent of those described in human multiple sclerosis (MS). These clusters were typically found adjacent to white matter lesions and their presence was associated with a chronic disease course. Extensive investigation of these clusters by histology did not identify features of lymphoid follicles, including organization of T and B cells into separate zones, CD35+ follicular dendritic cells (FDCs), or germinal centers (GCs). The majority of cluster B cells were IgD+ with little evidence of class switch. Consistent with this, B cells isolated from the spinal cord were of the naïve/memory CD38hi CD95lo phenotype. Nevertheless, they were CD62Llo and CD80hi compared to lymph node B cells suggesting that they were at least partly activated and primed to present antigen. Therefore, if meningeal B cells contribute to CNS pathology in autoimmunity, follicular differentiation is not necessary for the pathogenic mechanism.

Profound thrombocytopenia occurs in humans with sepsis and in mice administered lipopolysaccharide (LPS). Growing evidence indicates that platelets may contribute to these abnormalities, but whether that is a direct result of LPS activation of platelets or an indirect result of other inflammatory mechanisms remains unclear. Here we demonstrate that although platelets do not increase P-selectin expression in response to LPS, platelets bind more avidly to fibrinogen under flow conditions in a Toll-like receptor-4 (TLR4)-dependent manner. In addition, we find that CD41+ megakaryocytes grown from fetal livers and adult circulating platelets express significant amounts of TLR4. LPS induced thrombocytopenia in wild-type mice but not in TLR4-deficient (TLR4def) mice. Wild-type platelets accumulated in the lungs of wild-type mice in response to LPS; TLR4def platelets did not. However, wild-type platelets did not accumulate in the lungs of LPS-treated TLR4def mice. Neutrophils also accumulated in the lungs, and this preceded platelet accumulation. Neutrophil depletion completely abolished LPS-induced platelet sequestration into the lungs, but platelet depletion did not affect neutrophil accumulation. Thus, our data show for the first time that platelets do express functional levels of TLR4, which contribute to thrombocytopenia through neutrophil-dependent pulmonary sequestration in response to LPS. (Blood. 2005;106:2417-2423)

Signaling occurs between the liver and brain in cholestatic liver disease, giving rise to sickness behaviors such as fatigue. However, the signaling pathways involved are poorly defined. Circulating inflammatory mediator levels are increased in cholestasis, leading to speculation that they may be capable of activating circulating immune cells that subsequently could gain access to the brain. Indeed, we have identified that at day 10 after bile duct resection-induced cholestasis, there is activation of circulating monocytes that express tumor necrosis factor alpha (TNF-alpha) in conjunction with increased expression of adhesion molecules by cerebral endothelium. Moreover, using intravital microscopy, we have identified markedly enhanced leukocytes rolling along cerebral endothelial cells, mediated by P-selectin, in bile duct-resected (BDR) but not control mice. In addition, we have identified increased infiltration of monocytes (but not lymphocytes) into the brains of BDR mice and found that these infiltrating monocytes produce TNF-alpha. Furthermore, infiltration of TNF-alpha-secreting monocytes into the brains of cholestatic mice is associated with a broad activation of resident brain macrophages to produce TNF-alpha. In conclusion, cholestasis is associated with an activation of cerebral endothelium that recruits TNF-alpha-producing monocytes into the brain. We hypothesize that enhanced TNF-alpha release within the brain may contribute to the development of cholestasis-associated sickness behaviors, including fatigue.

Lymphocyte CD44 interactions with hyaluronan localized on the endothelium have been demonstrated to mediate rolling and regulate lymphocyte entry into sites of chronic inflammation. Because neutrophils also express CD44, we investigated the role of CD44 and hyaluronan in the multistep process of neutrophil recruitment. CD44−/− and wild-type control mice were intrascrotally injected with the neutrophil-activating chemokine, MIP-2, and leukocyte kinetics in the cremasteric microcirculation were investigated 4 h subsequently using intravital microscopy. Neither the rolling flux nor the rolling velocities were decreased in CD44−/− mice relative to wild-type mice. In vitro, neutrophils did not roll on the CD44 ligand hyaluronan, consistent with the in vivo data that CD44/hyaluronan did not mediate rolling. However, the number of adherent leukocytes in the venule was decreased by 65% in CD44−/− mice compared with wild-type mice. Leukocyte emigration was also greatly decreased in the CD44−/− mice. The same decrease in adhesion and emigration was observed in the wild-type mice given hyaluronidase. Histology revealed neutrophils as being the dominant infiltrating population. We generated chimeric mice that express CD44 either on their leukocytes or on their endothelium and found that CD44 on both the endothelium and neutrophils was important for optimal leukocyte recruitment into tissues. Of those neutrophils that emigrated in wild-type and CD44−/− mice, there was no impairment in migration through the interstitium. This study suggests that CD44 can mediate some neutrophil adhesion and emigration, but does not appear to affect subsequent migration within tissues.

Environmental factors strongly influence the development of autoimmune diseases, including multiple sclerosis. Despite this clear association, the mechanisms through which environment mediates its effects on disease are poorly understood. Pertussis toxin (PTX) functions as a surrogate for environmental factors to induce animal models of autoimmunity, such as experimental autoimmune encephalomyelitis. Although very little is known about the molecular mechanisms behind its function in disease development, PTX has been hypothesized to facilitate immune cell entry to the CNS by increasing permeability across the blood-brain barrier. Using intravital microscopy of the murine cerebromicrovasculature, we demonstrate that PTX alone induces the recruitment of leukocytes and of active T cells to the CNS. P-selectin expression was induced by PTX, and leukocyte/endothelial interactions could be blocked with a P-selectin-blocking Ab. P-selectin blockade also prevented PTX-induced increase in permeability across the blood-brain barrier. Therefore, permeability is a secondary result of recruitment, rather than the primary mechanism by which PTX induces disease. Most importantly, we show that PTX induces intracellular signals through TLR4, a receptor intimately associated with innate immune mechanisms. We demonstrate that PTX-induced leukocyte recruitment is dependent on TLR4 and give evidence that the disease-inducing mechanisms initiated by PTX are also at least partly dependent on TLR4. We propose that this innate immune pathway is a novel mechanism through which environment can initiate autoimmune disease of the CNS.

Experimental autoimmune encephalomyelitis (EAE) is mediated by inflammatory cells recruited from the circulation to the CNS. We used intravital microscopy to investigate the mechanisms of this recruitment. No leukocyte rolling and very little adhesion was observed in healthy control mice. In contrast, both rolling and adhesion was observed in brain postcapillary venules before onset of physical symptoms of EAE. Rolling and adhesion remained elevated for 2 wk and returned to near normal levels by 5 wk postsymptom onset. Consistent with a role for P-selectin in recruitment to the CNS, P-selectin protein was detected in the brains and spinal cords of EAE mice. Expression was highest before symptom onset and decreased over the next 2 wk. The importance of α4 integrin increased with time as anti-α4 integrin blocked ∼20, 50, and 60% of leukocyte rolling 2 days before disease onset, 5 days and 2 wk postonset of symptoms, respectively, and 85% of rolling 5 wk postsymptoms. Addition of anti-P-selectin to α4 integrin Ab-treated mice blocked all remaining rolling at each time point. Interestingly, however, α4 integrin-mediated rolling appeared to be entirely dependent on P-selectin as anti-P-selectin alone was able to completely block all leukocyte rolling. In the absence of rolling (with P-selectin Ab), a 70% reduction in adhesion was noted. A very similar reduction was seen when mice were treated with α4 integrin-blocking Ab. In conclusion, we describe increased leukocyte trafficking in the brains of EAE mice with important overlapping roles for both P-selectin and α4 integrin in mediating leukocyte-endothelial cell interactions.

We develop a new fusion protein reagent (MOG tag ), based on the extracellular domain of mouse myelin oligodendrocyte glycoprotein (MOG 1–125 ), designed to induce autoimmune responses in mice that incorporates both T and B cell recognition of antigen. Reports of similar reagents, primarily based on foreign MOG proteins, rely largely on disease incidence and severity, with little analysis of the underlying immune response or pathology. We characterize the immune response and central nervous system autoimmune disease elicited by MOG tag in mice and find that it results in the formation of a T cell-dependent germinal center B cell response. Unlike immunization with the short MOG 35–55 peptide, this response incorporated B cells able to recognize MOG protein. The autoimmune disease resulting from immunization with MOG tag was chronic with clear evidence of an ongoing immune response and active white and gray matter infiltration by T cells as well as formation of B cell clusters in the meninges. Interestingly, development of B cell clusters was not absolutely dependent on the ability of B cells to recognize MOG protein, as they were also present in mice immunized with short peptide and in mice with a mutant B cell receptor specific for an irrelevant antigen.

Gamma interferon (IFN-γ) plays a critical role in the early eradication of Anaplasma phagocytophilum . However, the mechanisms that regulate IFN-γ production upon infection remain poorly understood. Here we show that c-Jun NH 2 -terminal kinase 2 (JNK2) inhibits IFN-γ production during A. phagocytophilum infection. jnk2 -null mice were more refractory to infection with A. phagocytophilum and produced increased levels of IFN-γ after challenge with the pathogen. The resistance of jnk2 -null mice to A. phagocytophilum infection was due to elevated levels of IFN-γ secreted by conventional and natural killer (NK) T cells. The administration of α-galactosylceramide, a strong NK T-cell agonist, increased IFN-γ release and protected mice from A. phagocytophilum , further demonstrating the inhibitory effect of JNK2 on IFN-γ production. Collectively, these findings provide strong evidence that JNK2 is an important regulatory protein for IFN-γ secretion upon challenge with A. phagocytophilum .

There has been a great deal of interest in adhesion molecules as targets for the treatment of multiple sclerosis and other inflammatory diseases. In this study, we systematically evaluate α4 integrin and P-selectin as targets for therapy in murine models of multiple sclerosis–for the first time directly measuring the ability of their blockade to inhibit recruitment and relate this to clinical efficacy. Experimental autoimmune encephalomyelitis was induced in C57BL/6 or SJL/J mice and intravital microscopy was used to quantify leukocyte interactions within the CNS microvasculature. In both strains, pretreatment with blocking Abs to either α4 integrin or P-selectin reduced firm adhesion to a similar extent, but did not block it completely. The combination of the Abs was more effective than either Ab alone, although the degree of improvement was more evident in SJL/J mice. Similarly, dual blockade was much more effective at preventing the subsequent accumulation of fluorescently labeled leukocytes in the tissue in both strains. Despite evidence of blockade of leukocyte recruitment mechanisms, no clinical benefit was observed with anti-adhesion molecule treatments or genetic deletion of P-selectin in the C57BL/6 model, or in a pertussis toxin-modified model in SJL/J mice. In contrast, Abs to α4 integrin resulted in a significant delay in the onset of clinical signs of disease in the standard SJL/J model. Despite evidence of a similar ability to block firm adhesion, Abs to P-selectin had no effect. Importantly, combined blockade of both adhesion molecules resulted in significantly better clinical outcome than anti-α4 integrin alone.

The recruitment of leukocytes to a tissue is a critical step in the inflammatory response. Toll-like receptor 4 (TLR4) is an important receptor involved in the initiation of inflammatory responses. Administration of the ligand for TLR4, lipopolysaccharide, is often used to model inflammation—local responses to stimuli within a specific tissue and systemic responses such as those observed during endotoxic or septic shock. Here, we review work, which demonstrates that in response to local activation of TLR4, highly coordinated and multistep processes are initiated, ultimately resulting in the leukocyte's arrival at the inflamed tissue. In contrast, systemic activation of TLR4 results in nonspecific accumulation of leukocytes within the lung capillaries and liver sinusoids through mechanisms profoundly different than those involved in local tissue recruitment. Contrary to current dogma, leukocyte accumulation in the lung is dependent on endothelial rather than leukocyte activation. Finally, we discuss recent evidence suggesting that activation of leukocytes through TLR4, although still in the circulation, effectively paralyzes inflammatory cells, rendering them incapable of appropriate trafficking to inflamed tissues.

Fractalkine is a unique chemokine possessing a long mucin-like stalk and a transmembrane region that has been proposed to act as an adhesion molecule. We investigated the ability of fractalkine to recruit leukocytes from whole blood, using an immobilized fractalkine fusion protein in the parallel-plate flow-chamber assay. Significant adhesion of leukocytes to fractalkine peaked at 2 dynes/cm(2) but was minimal at 10 dynes/cm(2). In contrast, VCAM-1 could recruit cells from whole blood at 10 dynes/cm(2). Co-immobilization of fractalkine and VCAM-1 at 10 dynes/cm(2) resulted in a twofold increase in adherent cells compared with VCAM-1 alone, suggesting that fractalkine can mediate adhesion at high shear if combined with a molecule that can mediate leukocyte tethering. Pretreatment of blood with pertussis toxin eliminated this increase in adhesion, implicating intracellular signaling in fractalkine-mediated mechanisms of adhesion to co-immobilized fractalkine/VCAM-1. Analysis of the cell types recruited to fractalkine alone at low shear, or to fractalkine and VCAM-1 at 10 dynes/cm(2), revealed that monocytes were recruited to fractalkine with the highest specificity. In conclusion, fractalkine is unlikely to act alone at shear forces found in most vascular beds where it most likely co-operates with tethering molecules, e.g. VCAM-1, in the recruitment of monocytes.

Oncostatin M (OSM), a member of the IL-6 family has been postulated to be a potent recruiter of leukocytes, however information regarding the molecular mechanism(s) underlying this event is extremely limited. Therefore, the aim of this study was to investigate the role of OSM-mediated leukocyte recruitment in a human system in vitro under flow conditions. A parallel-plate flow chamber assay was used to examine leukocyte recruitment from whole blood by human umbilical vein endothelium treated for 24 hours with OSM. OSM in a dose-response manner revealed very significant leukocyte rolling and adhesion reaching optimal levels at a very low concentration of OSM (10 ng/ml). The OSM-induced leukocyte rolling and adhesion was comparable to levels seen with tumor necrosis factor. OSM was extremely selective for neutrophil recruitment (96%) with3% lymphocyte recruitment. By contrast, tumor necrosis factor-alpha revealed no such selectivity, recruiting 70% neutrophils and at least 25% lymphocytes and detectable levels of eosinophils at 24 hours. The molecular mechanism underlying the leukocyte recruitment seemed to be entirely dependent on P-selectin as leukocyte recruitment could be completely blocked by the addition of a P-selectin-blocking antibody. An elevation in both P-selectin message and protein was observed with 24 hours of OSM stimulation of endothelium. By contrast, E-selectin and VCAM-1 were not detectable after OSM stimulation. Similar results were seen with passaged dermal microvascular endothelium that does not have a prestored pool of P-selectin. Based on these results, we conclude that OSM may be a very selective potent recruiter of neutrophils in more prolonged inflammatory conditions, an event exclusively dependent on P-selectin.

Intravital microscopy has done much to elucidate the cascade of events involved in the recruitment of leukocytes to sites of inflammation. Here we review the physiological relevance of leukocyte rolling and some of the important subtleties of this process, highlighting limitations in our knowledge and directions for future investigation.

Contact sensitivity (CS) to haptens is related to delayed type hypersensitivity (DTH) and is a well characterized prototype of T cell mediated inflammation. CS is additionally dependent on a parallel immune pathway. In response to sensitization, a population of B-1 cells produces antigen-specific IgM which mediates a rapid inflammatory response to hapten re-exposure. Interference with this pathway prevents the full development of the classic delayed response and is therefore termed the “CS-Initiation” pathway. The identity of the B-1 cell subset responsible for CS-Initiation is not known. Here, we show that CS-Initiation is dependent on activation induced deaminase (AID), an enzyme central to the process of somatic hypermutation (SHM). Using adoptive transfer experiments, we demonstrate that the defect is specific to B-1 cells as transfer of wild type B-1 cells reconstitutes CS-Initiation in deficient recipients. We went on to identify and characterize a unique subpopulation of B-1 cells in the spleens of sensitized mice that possess initiation activity. Detailed analysis of B cell receptor genes isolated from these cells revealed evidence of AID-mediated SHM. The sensitivity of CS-Initiation to very low amounts of hapten and the dependence on AID suggests that these cells are selected to generate antibodies capable of mediating CS-Initiation. This work funded by the Canadian Institutes of Health Research, the American Academy of Allergy, Asthma and Immunology and the National Institutes of Health.

Summary We identify the interfollicular (IF) zone as the site where germinal center B cell and T follicular helper (Tfh) cell differentiation initiates. For the first 2 days postimmunization, antigen-specific T and B cells remained confined within the IF zone, formed long-lived interactions, and upregulated the transcriptional repressor Bcl6. T cells also acquired the Tfh cell markers CXCR5, PD-1, and GL7. Responding B and T cells migrated to the follicle interior directly from the IF zone, T cell immigration preceding B cells by 1 day. Notably, in the absence of cognate B cells, Tfh cells still formed and migrated to the follicle. However, without such B cells, PD-1, ICOS, and GL7 were no longer expressed on follicular Bcl6 hi T cells that nevertheless persisted in the follicle. Thus, Ag-specific B cells are required for the maintenance of the PD-1 hi ICOS hi GL7 hi Tfh cell phenotype within the follicle, but not for their initial differentiation in the IF zone.