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Alternative (M2)-polarized macrophages possess high capacities to produce specialized proresolving mediators (SPM; i.e., resolvins, protectins, and maresins) that play key roles in resolution of inflammation and tissue regeneration. Vacuolar (H + )-ATPase (V-ATPase) is fundamental in inflammatory cytokine trafficking and secretion and was implicated in macrophage polarization toward the M2 phenotype, but its role in SPM production and lipid mediator biosynthesis in general is elusive. In this study, we show that V-ATPase activity is required for the induction of SPM-biosynthetic pathways in human M2-like monocyte-derived macrophages (MDM) and consequently for resolution of inflammation. Blockade of V-ATPase by archazolid during IL-4-induced human M2 polarization abrogated 15-lipoxygenase-1 expression and prevented the related biosynthesis of SPM in response to pathogenic Escherichia coli , assessed by targeted liquid chromatography-tandem mass spectrometry-based metabololipidomics. In classically activated proinflammatory M1-like MDM, however, the biosynthetic machinery for lipid mediator formation was independent of V-ATPase activity. Targeting V-ATPase in M2 influenced neither IL-4-triggered JAK/STAT6 nor the mTOR complex 1 signaling but strongly suppressed the ERK-1/2 pathway. Accordingly, the ERK-1/2 pathway contributes to 15-lipoxygenase-1 expression and SPM formation in M2-like MDM. Targeting V-ATPase in vivo delayed resolution of zymosan-induced murine peritonitis accompanied by decreased SPM levels without affecting proinflammatory leukotrienes or PGs. Together, our data propose that V-ATPase regulates 15-lipoxygenase-1 expression and consequent SPM biosynthesis involving ERK-1/2 during M2 polarization, implying a crucial role for V-ATPase in the resolution of inflammation., (Copyright © 2019 by The American Association of Immunologists, Inc.)

IL-33 is an IL-1 cytokine superfamily member. Binding of IL-33 to the IL-33R induces activation of the canonical NF-κB signaling and activation of MAPKs. In bone marrow-derived dendritic cells, IL-33 induces the production of IL-6, IL-13, and TNF-α. However, the signaling pathways resulting in IL-33-induced effector functions of dendritic cells are unknown. In this article, we show that the IL-33-induced cytokine production is only partly dependent on p65. Thereby, p65 mediates the production of IL-6, but not of IL-13, whereas the p38-Mapk-activated protein kinases 2/3 (MK2/3) signaling module mediates the IL-13, but not the IL-6, production. In addition, GM-CSF, which is critical for the differentiation and proliferation of bone marrow-derived dendritic cells, potentiates the p65-dependent IL-6 and the p38-MK2/3-dependent IL-13 production. Furthermore, we found that effective TNF-α production is only induced in the presence of GM-CSF and IL-33 via the p38-MK2/3 signaling module. Taken together, we found that the p38-MK2/3 signaling module is essential to mediate IL-33-induced cytokine production in dendritic cells., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The neurobeachin-like 2 protein (Nbeal2) belongs to the family of beige and Chediak-Higashi (BEACH) domain proteins. Loss-of-function mutations in the human NBEAL2 gene or Nbeal2 deficiency in mice cause gray platelet syndrome, a bleeding disorder characterized by macrothrombocytopenia, splenomegaly, and paucity of α-granules in megakaryocytes and platelets. We found that in mast cells, Nbeal2 regulates the activation of the Shp1-STAT5 signaling axis and the composition of the c-Kit/STAT signalosome. Furthermore, Nbeal2 mediates granule formation and restricts the expression of the transcription factors, IRF8, GATA2, and MITF as well as of the cell-cycle inhibitor p27, which are essential for mast cell differentiation, proliferation, and cytokine production. These data demonstrate the relevance of Nbeal2 in mast cells above and beyond granule biosynthesis., (Copyright © 2017 by The American Association of Immunologists, Inc.)

The IL-1R family member IL-33R mediates Fcε-receptor-I (FcεRI)-independent activation of mast cells leading to NF-κB activation and consequently the production of cytokines. IL-33 also induces the activation of MAPKs, such as p38. We aimed to define the relevance of the p38-targets, the MAPK-activated protein kinases 2 and 3 (MK2 and MK3) in IL-33-induced signaling and the resulting mast cell effector functions in vitro and in vivo. We demonstrate that the IL-33-induced IL-6 and IL-13 production strongly depends on the MK2/3-mediated activation of ERK1/2 and PI3K signaling. Furthermore, in the presence of the stem cell factors, IL-33 did induce an MK2/3-, ERK1/2- and PI3K-dependent production of TNF-α. In vivo, the loss of MK2/3 in mast cells decreased the IL-33-induced leukocyte recruitment and the resulting skin inflammation. Therefore, the MK2/3-dependent signaling in mast cells is essential to mediate IL-33-induced inflammatory responses. Thus, MK2/3 are potential therapeutic targets for suppression of IL-33-induced inflammation skin diseases such as psoriasis., (Copyright © 2016 by The American Association of Immunologists, Inc.)

IL-33 is a new member of the IL-1 family, which plays a crucial role in inflammatory response, enhancing the differentiation of dendritic cells and alternatively activated macrophages (AAM). Based on the evidence of IL-33 expression in bone, we hypothesized that IL-33 may shift the balance from osteoclast to AAM differentiation and protect from inflammatory bone loss. Using transgenic mice overexpressing human TNF, which develop spontaneous joint inflammation and cartilage destruction, we show that administration of IL-33 or an IL-33R (ST2L) agonistic Ab inhibited cartilage destruction, systemic bone loss, and osteoclast differentiation. Reconstitution of irradiated hTNFtg mice with ST2(-/-) bone marrow led to more bone loss compared with the chimeras with ST2(+/+) bone marrow, demonstrating an important endogenous role of the IL-33/ST2L pathway in bone turnover. The protective effect of IL-33 on bone was accompanied by a significant increase of antiosteoclastogenic cytokines (GM-CSF, IL-4, and IFN-γ) in the serum. In vitro IL-33 directly inhibits mouse and human M-CSF/receptor activator for NF-κB ligand-driven osteoclast differentiation. IL-33 acts directly on murine osteoclast precursors, shifting their differentiation toward CD206(+) AAMs via GM-CSF in an autocrine fashion. Thus, we show in this study that IL-33 is an important bone-protecting cytokine and may be of therapeutic benefit in treating bone resorption.

Reactive oxygen species (ROS) are important in the immune defense against invading pathogens, but they are also key molecules in the regulation of inflammatory reactions. Low levels of ROS production due to a polymorphism in the neutrophil cytosolic factor 1 (Ncf1) gene are associated with autoimmunity and arthritis severity in mouse models induced with adjuvant. We established an adjuvant-free arthritis model in which disease is induced by injection of the autoantigen collagen type II (CII) and depends on IL-5-producing T cells and eosinophils. In addition, the transgenic expression of mutated mouse CII allowed us to investigate an autoreactive immune response to an autologous Ag and by that natural tolerance mechanism. We show that a deficient ROS production, due to a spontaneous mutation in Ncf1, leads to increased autoantibody production and expansion of IL-33R-expressing T cells, impaired T cell tolerance toward tissue-specific CII, and severe arthritis in this unique model without disturbing adjuvant effects. These results demonstrate that the insufficient production of ROS promotes the breakdown of immune tolerance and development of autoimmune and adjuvant-free arthritis through an IL-5- and IL33R-dependent T cell activation pathway.

EBV is a candidate trigger of rheumatoid arthritis (RA). We determined both EBV-specific T cell and B cell responses and cell-associated EBV DNA copies in patients with RA and demographically matched healthy virus carriers. Patients with RA showed increased and broadened IgG responses to lytic and latent EBV-encoded Ags and 7-fold higher levels of EBV copy numbers in circulating blood cells. Additionally, patients with RA exhibited substantial expansions of CD8(+) T cells specific for pooled EBV Ags expressed during both B cell transformation and productive viral replication and the frequency of CD8(+) T cells specific for these Ags correlated with cellular EBV copy numbers. In contrast, CD4(+) T cell responses to EBV and T cell responses to human CMV Ags were unchanged, altogether arguing against a defective control of latent EBV infection in RA. Our data show that the regulation of EBV infection is perturbed in RA and suggest that increased EBV-specific effector T cell and Ab responses are driven by an elevated EBV load in RA.

Mycobacterium tuberculosis remains one of the top microbial killers of humans causing approximately 2 million deaths annually. More than 90% of the 2 billion individuals infected never develop active disease, indicating that the immune system is able to generate mechanisms that control infection. However, the immune response generally fails to achieve sterile clearance of bacilli. Using adoptive cell transfer into C57BL/6J-Rag1(tm1Mom) mice (Rag1(-/-)), we show that regulatory T cells prevent eradication of tubercle bacilli by suppressing an otherwise efficient CD4+ T cell response. This protective CD4+ T cell response was not correlated with increased numbers of IFN-gamma- or TNF-alpha-expressing cells or general expression levels of IFN-gamma or inducible NO synthase in infected organs compared with wild-type C57BL/6 animals. Furthermore, suppression of protection by cotransferred regulatory T cells was neither accompanied by a general increase of IL-10 expression nor by higher numbers of IL-10-producing CD4+ T cells.

Th1- and Th2-polarized immune responses are crucial in the defense against pathogens but can also promote autoimmunity and allergy. The chemokine receptors CXCR3 and CCR4 have been implicated in differential trafficking of IFN-gamma- and IL-4-producing T cells, respectively, but also in tissue and inflammation-specific homing independent of cytokine responses. Here, we tested whether CD4+ T cells isolated from murine tissues under homeostatic or inflammatory conditions exhibit restricted patterns of chemotactic responses that correlate with their production of IFN-gamma, IL-4, or IL-10. In uninfected mice, IL-4-producing T cells preferentially migrated to the CCR4 ligand, CCL17, whereas IFN-gamma-expressing T cells as well as populations of IL-4+ or IL-10+ T cells migrated to the CXCR3 ligand, CXCL9. All cytokine-producing T cell subsets strongly migrated to the CXCR4 ligand, CXCL12. We assessed chemotaxis of T cells isolated from mice infected with influenza A virus or the nematode Nippostrongylus brasiliensis, which induce a strong Th1 or Th2 response in the lung, respectively. Unexpectedly, the chemotactic responses of IL-4+ T cells and T cells expressing the immunosuppressive cytokine IL-10 were influenced not only by the strongly Th1- or Th2-polarized environments but also by their anatomical localization, i.e., lung or spleen. In contrast, IFN-gamma+ T cells exhibited robust chemotaxis toward CXCL9 and had the most consistent migration pattern in both infection models. The results support a model in which the trafficking responses of many effector and regulatory T cells are regulated as a function of the infectious and tissue environments.

Infections sometimes associate with exacerbations of autoimmune diseases through pathways that are poorly understood. Ag-specific mechanisms such as cross-reactivity between a microbial Ag and a self-Ag have received no direct support. In this study, we show that injection of LPS induces experimental autoimmune encephalomyelitis in TCR-transgenic mice and relapse of encephalomyelitis in normal mice. This form of treatment induces proliferation and cytokine production in a fraction of effector/memory Th lymphocytes in vitro via physical contact of Th cells with CD4(-) LPS-responsive cells. TCR-mediated signals are not necessary; rather what is required is ligation of costimulatory receptors on Th cells by costimulatory molecules on the CD4(-) cells. This form of bystander activation provides an Ag-independent link between infection and autoimmunity that might fit the clinical and epidemiological data on the connection between infection and autoimmunity better than the Ag-specific models.

Rheumatoid arthritis is a chronic inflammatory disease associated with certain HLA-DR4 subtypes. The target autoantigen(s) is unknown, but type II collagen (CII) is a candidate, with a single immunodominant DR4-restricted 261-273 T cell epitope (CII(261-273)). In the present study, we have prepared HLA-DR4:CII(261-273) tetramers and analyzed peripheral blood, lymph node, and synovial fluid cells from DR4-transgenic mice with early and late collagen-induced arthritis to draw a fuller picture of the role of CII-reactive Th cells in disease development. Their frequencies increased approximately 20-fold in blood 1-2 wk postimmunization, and even more in acutely arthritic joints. Our data strongly suggest that CII-specific Th cells are necessary, but not sufficient for collagen-induced arthritis. The CII-specific Th cells displayed an activated proinflammatory Th1 phenotype, and their expansion correlated with onset and severity of arthritis and also with anti-CII Ab levels. Surprisingly, shortly after the first clinical signs of arthritis, activated HLA-DR4:CII tetramer(+) cells became undetectable in the synovial fluid and rare in the blood, but persisted in lymph nodes. Consequently, future human studies should focus on patients with early arthritis, and on their synovial cells, to re-evaluate the occurrence and pathogenic importance of CII-specific or other Th cells in rheumatoid arthritis.

Rheumatoid arthritis is a chronic inflammatory disease primarily affecting the joints. The search for arthritogenic autoantigens that trigger autoimmune responses in rheumatoid arthritis has largely focused on cartilage- or joint-specific Ags. In this study, we show that immunization with the ubiquitously expressed glycolytic enzyme glucose-6-phosphate isomerase (G6PI) induces severe peripheral symmetric polyarthritis in normal mice. In genetically unaltered mice, T cells are indispensable for both the induction and the effector phase of G6PI-induced arthritis. Arthritis is cured by depletion of CD4(+) cells. In contrast, Abs and FcgammaR(+) effector cells are necessary but not sufficient for G6PI-induced arthritis in genetically unaltered mice. Thus, the complex pathogenesis of G6PI-induced arthritis in normal mice differs strongly from the spontaneously occurring arthritis in the transgenic K/B x N model where Abs against G6PI alone suffice to induce the disease. G6PI-induced arthritis demonstrates for the first time the induction of organ-specific disease by systemic autoimmunity in genetically unaltered mice. Both the induction and effector phase of arthritis induced by a systemic autoimmune response can be dissected and preventive and therapeutic strategies evaluated in this model.

The immune response against the intracellular bacterium Listeria monocytogenes involves both CD4(+) and CD8(+) T cells. We used the MHC class II-presented peptide listeriolysin(189-201) to characterize the organ-specific CD4(+) T cell response during infection. Systemic listeriosis resulted in a strong peptide-specific CD4(+) T cell response with frequencies of 1/100 and 1/30 CD4(+) splenocytes at the peak of primary and secondary response, respectively. This response was not restricted to lymphoid organs, because we detected specific CD4(+) T cells in all tissues analyzed. However, the tissue distribution of the T cell response was dependent on the route of infection. After i.v. infection, the strongest CD4(+) T cell response and the highest levels of memory cells were observed in spleen and liver, the major sites of L. monocytogenes replication. After oral infection, we detected a strong response in the liver, the lamina propria, and the intestinal epithelium. These tissues also harbored the highest frequencies of listeriolysin(189-201)-specific CD4(+) memory T cells 5-8 wk post oral infection. Our results show that kinetics and magnitude of the CD4(+) T cell response and the accumulation of CD4(+) memory T cells depend on the route of infection and are regulated in a tissue-specific way.

TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces apoptosis in susceptible cells, which can be both malignant and nontransformed. Despite homologies among the death ligands, there are great differences between the TRAIL system on the one hand and the TNF and CD95 systems on the other hand. In particular, TRAIL-induced apoptosis differs between rodents and man. Studies on animal models of autoimmune diseases suggested an influence of TRAIL on T cell growth and effector functions. Because we previously demonstrated that TRAIL does not induce apoptosis in human (auto)antigen-specific T cells, we now asked whether TRAIL exhibits other immunoregulatory properties in these cells. Active TRAIL inhibited calcium influx through store-operated calcium release-activated calcium channels, IFN-gamma/IL-4 production, and proliferation. These effects were independent of APC, Ag specificity, and Th differentiation, and no differences were detected between healthy donors and multiple sclerosis patients. TRAIL affected neither the expression of the cell cycling inhibitor p27(Kip1) nor the capacity of T cells to produce IL-2 upon Ag rechallenge, indicating that signaling via TRAIL receptor does not induce T cell anergy. Instead, the TRAIL-induced hypoproliferation could be attributed to the down-regulation of the cyclin-dependent kinase 4, indicating a G(1) arrest of the cell cycle. Thus, although it does not contribute to mechanisms of peripheral T cell tolerance such as clonal anergy or deletion by apoptosis, TRAIL can directly inhibit activation of human T cells via blockade of calcium influx.

The orphan receptor T1/ST2, a member of the IL-1R family, is preferentially expressed on the surface of murine Th2 cells. In this study, we analyzed the kinetics and function of T1/ST2 expression on Th2 cells in vitro. Whereas naive CD4(+) cells did not express T1/ST2, most CD4(+) cells became T1/ST2(+) upon repeated antigenic stimulation under Th2-polarizing conditions. Flow cytometric analyses revealed that the kinetics of T1/ST2 expression on Th2 cells was delayed compared with the kinetics of type 2 cytokine production. Exogenous IL-6, IL-5, IL-1, and TNF-alpha enhanced the expression of T1/ST2 on Th2 cells, and IL-6 was by far most effective in this regard. However, the expression of T1/ST2 did not depend on the presence of IL-6 and was also detected in IL-6-deficient mice. Most important, cross-linking of T1/ST2 provided a costimulatory signal for Th2 but not Th1 cells and directly induced proliferation and type 2 cytokine production. Thus, T1/ST2 is not only a Th2 cell marker but also plays an important role in the activation of Th2 cells.

Naive Th cells can be directed in vitro to develop into Th1 or Th2 cells by IL-12 or IL-4, respectively. In vivo, chronic immune reactions lead to polarized Th cytokine patterns. We found earlier that Borrelia burgdorferi, the spirochaete that causes Lyme disease, induces Th1 development in alpha beta TCR-transgenic Th cells. Here, we used TCR-transgenic Th cells and oligonucleotide arrays to analyze the differences between Th1 cells induced by IL-12 vs those induced by B. burgdorferi. Transgenic Th cells primed with peptide in the presence of B. burgdorferi expressed several mRNAs, including the mRNA encoding IL-17, at significantly higher levels than Th cells primed with peptide and IL-12. Cytometric single-cell analysis of Th cell cytokine production revealed that IL-17 cannot be categorized as either Th1 or Th2 cytokine. Instead, almost all IL-17-producing Th cells simultaneously produced TNF-alpha and most IL-17(+) Th cells also produced GM-CSF. This pattern was also observed in humans. Th cells from synovial fluid of patients with Lyme arthritis coexpressed IL-17 and TNF-alpha upon polyclonal stimulation. The induction of IL-17 production in Th cells is not restricted to B. burgdorferi. Priming of TCR-transgenic Th cells in the presence of mycobacterial lysates also induced IL-17/TNF-alpha coproduction. The physiological stimulus for IL-17 production was hitherto unknown. We show here for the first time that microbial stimuli induce the expression of IL-17 together with TNF-alpha in both murine and human T cells. Chronic IL-17 expression induced by microbes could be an important mediator of infection-induced immunopathology.

Activation of autoreactive T cells is a crucial event in the pathogenesis of autoimmune diseases. Cross-reactivity between microbial and self Ags (molecular mimicry) is one hypothesis that could explain the activation of autoreactive T cells. We have systematically examined this hypothesis in experimental autoimmune encephalomyelitis using mice bearing exclusively myelin basic protein (MBP)-specific T cells (designated T+ alpha-). A peptide substitution analysis was performed in which each residue of the MBPAc1-11 peptide was exchanged by all 20 naturally occurring amino acids. This allowed the definition of the motif (supertope) that is recognized by the MBPAc1-11-specific T cells. The supertope was used to screen protein databases (SwissProt and TREMBL). By the search, 832 peptides of microbial origin were identified and synthesized. Of these, 61 peptides induced proliferation of the MBPAc1-11-specific transgenic T cells in vitro. Thus, the definition of a supertope by global amino acid substitution can identify multiple microbial mimic peptides that activate an encephalitogenic TCR. Peptides with only two native MBP-residues were sufficient to activate MBPAc1-11-specific T cells in vitro, and experimental autoimmune encephalomyelitis could be induced by immunizing mice with a mimic peptide with only four native MBP residues.

Th cells are categorized into subsets based on the cytokine production of in vitro-differentiated Th populations. For in vivo-differentiated Th subsets, little is known about the heterogeneity of cytokine production in single cells. We recently described a molecule, T1/ST2, that is preferentially expressed on the surface of Th2 cells. Here we combined high-gradient magnetic cell separation with four-color single-cell cytometry to analyze simultaneously three intracellular cytokines and T1/ST2 surface expression on CD4+ cells from lungs containing granulomas induced by Schistosoma mansoni eggs. T1/ST2 was highly up-regulated on CD4+ T cells from hepatic granulomas and granulomatous lungs. T1/ST2+ cells from granulomatous lungs preferentially produced type 2 cytokines ex vivo. In the total CD4+ population, coexpression of type 1 and type 2 cytokines occurred frequently. However, such coproduction was drastically reduced in T1/ST2+ cells compared with T1/ST2- cells. Coexpression of type 1 and type 2 cytokines was also rare in cells simultaneously producing two cytokines of one type. These findings indicate that individual CD4+ T cells in vivo have different levels of commitment to a certain Th phenotype. Coexpression of two type 2 cytokines or production of one type 2 cytokine together with surface expression of T1/ST2 indicate advanced commitment to the Th2 phenotype.

In a murine model of T cell-mediated autoimmune disease, experimental autoimmune encephalitis (EAE), 80% of all encephalitogenic T cell clones in H-2u mice use the V beta 8.2 TCR element. To induce EAE in susceptible strains of mice either heat-killed Bordetella pertussis organisms or Bordetella pertussis toxin (PT) must be injected in addition to Ag in CFA. We investigated the mechanisms by which PT facilitates the induction of EAE. Our data show, that PT interferes with the induction of Ag-induced peripheral T cell anergy. Furthermore it has a specific adjuvanticity for the autoantigen pAc1-11 in vivo and acts as a selective mitogen in vitro. We also tested the hypothesis that PT is a bacterial superantigen that specifically expands the V beta 8.2+ subset of T cells, thereby expanding the encephalitogenic T cell clones that are contained in this subset, so that the number of autoreactive T cells is brought over a critical threshold, necessary to induce autoimmune disease. Our data show that PT is not a superantigen. Staphylococcal enterotoxin B, a V beta 8.2-specific superantigen, does not enhance the immune response to the encephalitogenic peptide.

We have used an approach of linking previously characterized T cell epitopes into immunologically complex synthetic peptides in order to investigate the mechanism of immunodominance. Our results show that first, cI12-26 is highly dominant following immunization with the lambda repressor (cI) protein, but is a minor epitope in the context of the cI:NP peptide. In contrast, the dominant epitope in response to the cI:NP peptide is a new junctional epitope, which is composed of sequences derived from both the cI and influenza nucleoprotein (NP) segments of the composite peptide. Second, T cell recognition of cI:NP is not significantly altered by Ag processing, based on results from glutaraldehyde-fixed APC. Third, the relative affinities of cI and cI:NP for MHC binding are similar, based on in vitro competition, excluding competition at the level of MHC binding as the determinant of immunodominance. Taken together, these results are consistent with the hypothesis that immunodominance of cI:NP is determined by peptide conformation, which affects the configuration of peptide binding to MHC, thus altering T cell recognition. In conclusion, immunodominance is not simply a function of the primary amino acid sequence, but is a function of the context of the epitope within the protein molecule.