CUX1 and IκBζ (NFKBIZ) mediate the synergistic inflammatory response to TNF and IL-17A in stromal fibroblasts.

The role of stromal fibroblasts in chronic inflammation is unfolding. In rheumatoid arthritis, leukocyte-derived cytokines TNF and IL-17A work together, activating fibroblasts to become a dominant source of the hallmark cytokine IL-6. However, IL-17A alone has minimal effect on fibroblasts. To identify key mediators of the synergistic response to TNF and IL-17A in human synovial fibroblasts, we performed time series, dose-response, and gene-silencing transcriptomics experiments. Here we show that in combination with TNF, IL-17A selectively induces a specific set of genes mediated by factors including cut-like homeobox 1 (CUX1) and IκBζ (NFKBIZ). In the promoters of CXCL1, CXCL2, and CXCL3, we found a putative CUX1-NF-κB binding motif not found elsewhere in the genome. CUX1 and NF-κB p65 mediate transcription of these genes independent of LIFR, STAT3, STAT4, and ELF3. Transcription of NFKBIZ, encoding the atypical IκB factor IκBζ, is IL-17A dose-dependent, and IκBζ only mediates the transcriptional response to TNF and IL-17A, but not to TNF alone. In fibroblasts, IL-17A response depends on CUX1 and IκBζ to engage the NF-κB complex to produce chemoattractants for neutrophil and monocyte recruitment.

Cell Intrinsic Deregulated ß-Catenin Signaling Promotes Expansion of Bone Marrow Derived Connective Tissue Type Mast Cells, Systemic Inflammation, and Colon Cancer.

Mast cells constitutively express ß-catenin and expand in solid tumors such as colon and skin cancer. However, the role of ß-catenin signaling in mast cells and the cause or effect of mast cell expansion and tumor growth has yet to be established. In earlier studies we used mast cell depletion and protease staining approaches, to provide evidence for a causative role of mast cells in small bowel polyposis, and related specific phenotypes and distributions of tumor infiltrating mast cells to stages of tumor growth. Here we report that, stabilization of ß-catenin expands mast cells to promote high incidence of colon polyposis and infrequent small bowel polyps and skin cancer. Expression of a dominant acting ß-catenin in mast cells (5CreCAT) stimulated maturation and expression of granule stored proteases. Both mucosal and connective tissue type mast cells accumulated in colonic small bowel polyps independent of gender, and mice developed chronic systemic inflammation with splenomegaly. Reconstitution of polyposis-prone mice with bone marrow from 5CreCAT mice resulted in focal expansion of connective tissue like mast cells, which are normally rare in benign polyps and characteristically expand during adenoma-to-carcinoma transition. Our findings highlight a hitherto unknown contribution of ß-catenin signaling in mast cells to their maturation and to increased risk of colon cancer.

PD-1hiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

Mouse Mast Cell Protease-4 Recruits Leukocytes in the Inflammatory Phase of Surgically Wounded Skin.

Objective: Mouse mast cell protease-4 (mMCP-4, also known as chymase) has both pro- and anti-inflammatory roles depending on the disease model. However, its effects have not been studied in surgically wounded skin. Given the significant clinical applications of modulating the inflammatory response in wound healing, we examined the role of mMCP-4 and the effect of its inhibitor chymostatin on leukocyte and polymorphonuclear cell (PMN) recruitment in our skin model. Approach: Recruitment was assessed on day-1 postwounding of three groups of mice (n = 10 each): mMCP-4 null mice, wild-type (WT) mice treated with the mMCP-4 inhibitor chymostatin, and WT with no other intervention. Leukocytes were stained with CD-45 cell marker, and PMN cells were stained with chloroacetate esterase. Results: The WT mice had 27 ± 9 leukocytes per field compared with 11 ± 6 for the mMCP-4 nulls, a decrease of 60% (p = 0.03), whereas the chymostatin-injected group had a count comparable with the uninjected WT controls at 24 ± 9. The WT group had a PMN count of 96 ± 12 cells, compared with just 24 ± 8 in the mMCP-4 null group, a decrease of 75% (p = 0.001), whereas the chymostatin-treated group had 60 ± 18 cells, a decrease of 38% compared with the WT group (p = 0.03). Innovation: We showed that the inflammatory process can be influenced by impeding the arrival of PMNs into the surgically injured site using the mMCP-4 inhibitor chymostatin. Conclusion: Chymase contributes to the recruitment of white blood cells in surgically wounded skin.

Deficiency of mouse mast cell protease 4 mitigates cardiac dysfunctions in mice after myocardium infarction.

Mouse mast cell protease-4 (mMCP4) is a chymase that has been implicated in cardiovascular diseases, including myocardial infarction (MI). This study tested a direct role of mMCP4 in mouse post-MI cardiac dysfunction and myocardial remodeling. Immunoblot and immunofluorescent double staining demonstrated mMCP4 expression in cardiomyocytes from the infarct zone from mouse heart at 28 day post-MI. At this time point, mMCP4-deficient Mcpt4-/- mice showed no difference in survival from wild-type (WT) control mice, yet demonstrated smaller infarct size, improved cardiac functions, reduced macrophage content but increased T-cell accumulation in the infarct region compared with those of WT littermates. mMCP4-deficiency also reduced cardiomyocyte apoptosis and expression of TGF-ß1, p-Smad2, and p-Smad3 in the infarct region, but did not affect collagen deposition or α-smooth muscle actin expression in the same area. Gelatin gel zymography and immunoblot analysis revealed reduced activities of matrix metalloproteinases and expression of cysteinyl cathepsins in the myocardium, macrophages, and T cells from Mcpt4-/- mice. Immunoblot analysis also found reduced p-Smad2 and p-Smad3 in the myocardium from Mcpt4-/- mice, yet fibroblasts from Mcpt4-/- mice showed comparable levels of p-Smad2 and p-Smad3 to those of WT fibroblasts. Flow cytometry, immunoblot analysis, and immunofluorescent staining demonstrated that mMCP4-deficiency reduced the expression of proapoptotic cathepsins in cardiomyocytes and protected cardiomyocytes from H2O2-induced apoptosis. This study established a role of mMCP4 in mouse post-MI dysfunction by regulating myocardial protease expression and cardiomyocyte death without significant impact on myocardial fibrosis or survival post-MI in mice.

Mixed-effects association of single cells identifies an expanded effector CD4+ T cell subset in rheumatoid arthritis.

High-dimensional single-cell analyses have improved the ability to resolve complex mixtures of cells from human disease samples; however, identifying disease-associated cell types or cell states in patient samples remains challenging because of technical and interindividual variation. Here, we present mixed-effects modeling of associations of single cells (MASC), a reverse single-cell association strategy for testing whether case-control status influences the membership of single cells in any of multiple cellular subsets while accounting for technical confounders and biological variation. Applying MASC to mass cytometry analyses of CD4+ T cells from the blood of rheumatoid arthritis (RA) patients and controls revealed a significantly expanded population of CD4+ T cells, identified as CD27- HLA-DR+ effector memory cells, in RA patients (odds ratio, 1.7; P = 1.1 × 10-3). The frequency of CD27- HLA-DR+ cells was similarly elevated in blood samples from a second RA patient cohort, and CD27- HLA-DR+ cell frequency decreased in RA patients who responded to immunosuppressive therapy. Mass cytometry and flow cytometry analyses indicated that CD27- HLA-DR+ cells were associated with RA (meta-analysis P = 2.3 × 10-4). Compared to peripheral blood, synovial fluid and synovial tissue samples from RA patients contained about fivefold higher frequencies of CD27- HLA-DR+ cells, which comprised ~10% of synovial CD4+ T cells. CD27- HLA-DR+ cells expressed a distinctive effector memory transcriptomic program with T helper 1 (TH1)- and cytotoxicity-associated features and produced abundant interferon-γ (IFN-γ) and granzyme A protein upon stimulation. We propose that MASC is a broadly applicable method to identify disease-associated cell populations in high-dimensional single-cell data.

Mast cells promote small bowel cancer in a tumor stage-specific and cytokine-dependent manner.

Mast cells (MCs) are tissue resident sentinels that mature and orchestrate inflammation in response to infection and allergy. While they are also frequently observed in tumors, the contribution of MCs to carcinogenesis remains unclear. Here, we show that sequential oncogenic events in gut epithelia expand different types of MCs in a temporal-, spatial-, and cytokine-dependent manner. The first wave of MCs expands focally in benign adenomatous polyps, which have elevated levels of IL-10, IL-13, and IL-33, and are rich in type-2 innate lymphoid cells (ILC2s). These vanguard MCs adhere to the transformed epithelial cells and express murine mast cell protease 2 (mMCP2; a typical mucosal MC protease) and, to a lesser extent, the connective tissue mast cell (CTMC) protease mMCP6. Persistence of MCs is strictly dependent on T cell-derived IL-10, and their loss in the absence of IL-10-expressing T cells markedly delays small bowel (SB) polyposis. MCs expand profusely in polyposis-prone mice when T cells overexpress IL-10. The frequency of polyp-associated MCs is unaltered in response to broad-spectrum antibiotics, arguing against a microbial component driving their recruitment. Intriguingly, when polyps become invasive, a second wave of mMCP5+/mMCP6+ CTMCs expands in the tumor stroma and at invasive tumor borders. Ablation of mMCP6 expression attenuates polyposis, but invasive properties of the remaining lesions remain intact. Our findings argue for a multistep process in SB carcinogenesis in which distinct MC subsets, and their elaborated proteases, guide disease progression.

Megakaryocytes compensate for Kit insufficiency in murine arthritis.

The growth factor receptor Kit is involved in hematopoietic and nonhematopoietic development. Mice bearing Kit defects lack mast cells; however, strains bearing different Kit alleles exhibit diverse phenotypes. Herein, we investigated factors underlying differential sensitivity to IgG-mediated arthritis in 2 mast cell-deficient murine lines: KitWsh/Wsh, which develops robust arthritis, and KitW/Wv, which does not. Reciprocal bone marrow transplantation between KitW/Wv and KitWsh/Wsh mice revealed that arthritis resistance reflects a hematopoietic defect in addition to mast cell deficiency. In KitW/Wv mice, restoration of susceptibility to IgG-mediated arthritis was neutrophil independent but required IL-1 and the platelet/megakaryocyte markers NF-E2 and glycoprotein VI. In KitW/Wv mice, platelets were present in numbers similar to those in WT animals and functionally intact, and transfer of WT platelets did not restore arthritis susceptibility. These data implicated a platelet-independent role for the megakaryocyte, a Kit-dependent lineage that is selectively deficient in KitW/Wv mice. Megakaryocytes secreted IL-1 directly and as a component of circulating microparticles, which activated synovial fibroblasts in an IL-1-dependent manner. Transfer of WT but not IL-1-deficient megakaryocytes restored arthritis susceptibility to KitW/Wv mice. These findings identify functional redundancy among Kit-dependent hematopoietic lineages and establish an unanticipated capacity of megakaryocytes to mediate IL-1-driven systemic inflammatory disease.

Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis.

CD4+ T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4+ T cells within affected tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1hiCXCR5-CD4+ T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1hiCXCR5- 'peripheral helper' T (TPH) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1hiCXCR5+ T follicular helper cells, TPH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between TPH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in TPH cells. TPH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.

Methods for the study of mast cell recruitment and accumulation in different tissues.

Mast cells (MC) are important effector cells involved in a wide range of inflammatory diseases. The lineage-committed, tissue-localized progenitor (MCp) is not easily identified histochemically like the mature MC because they lack the distinctive cytoplasmic granules. However, they can be identified by their unique cell surface phenotype and by their ability to be expanded in culture using selective growth factors. Here we describe the methods that allow evaluation of MCp and mature MC in peripheral tissues under basal and inflammatory conditions. Thus, one can enumerate mature MC as well as immature committed progenitors in order to study basal homing, inflammatory recruitment, maturation, and life span. We also provide an analysis of difficulties that could emerge during these procedures.

Mouse mast cell protease-6 and MHC are involved in the development of experimental asthma.

Allergic asthma is a complex disease with a strong genetic component where mast cells play a major role by the release of proinflammatory mediators. In the mouse, mast cell protease-6 (mMCP-6) closely resembles the human version of mast cell tryptase, ß-tryptase. The gene that encodes mMCP-6, Tpsb2, resides close by the H-2 complex (MHC gene) on chromosome 17. Thus, when the original mMCP-6 knockout mice were backcrossed to the BALB/c strain, these mice were carrying the 129/Sv haplotype of MHC (mMCP-6(-/-)/H-2bc). Further backcrossing yielded mMCP-6(-/-) mice with the BALB/c MHC locus. BALB/c mice were compared with mMCP-6(-/-) and mMCP-6(-/-)/H-2bc mice in a mouse model of experimental asthma. Although OVA-sensitized and challenged wild type mice displayed a striking airway hyperresponsiveness (AHR), mMCP-6(-/-) mice had less AHR that was comparable with that of mMCP-6(-/-)/H-2bc mice, suggesting that mMCP-6 is required for a full-blown AHR. The mMCP-6(-/-)/H-2bc mice had strikingly reduced lung inflammation, IgE responses, and Th2 cell responses upon sensitization and challenge, whereas the mMCP-6(-/-) mice responded similarly to the wild type mice but with a minor decrease in bronchoalveolar lavage eosinophils. These findings suggest that inflammatory Th2 responses are highly dependent on the MHC-haplotype and that they can develop essentially independently of mMCP-6, whereas mMCP-6 plays a key role in the development of AHR.

IL-33/ST2 axis promotes mast cell survival via BCLXL.

Mast cells (MC) are potent innate immune cells that accumulate in chronically inflamed tissues. MC express the IL-33 receptor IL-1 receptor-related protein ST2 at high level, and this IL-1 family cytokine both activates MC directly and primes them to respond to other proinflammatory signals. Whether IL-33 and ST2 play a role in MC survival remains to be defined. In skin-derived human MC, we found that IL-33 attenuated MC apoptosis without altering proliferation, an effect mediated principally through the antiapoptotic molecule B-cell lymphoma-X large (BCLXL). Murine MC demonstrated a similar mechanism, dependent entirely on ST2. In line with these observations, St2(-/-) mice exhibited reduced numbers of tissue MC in inflamed arthritic joints, in helminth-infected intestine, and in normal peritoneum. To confirm an MC-intrinsic role for ST2 in vivo, we performed peritoneal transfer of WT and St2(-/-) MC. In St2(-/-) hosts treated with IL-33 and in WT hosts subjected to thioglycollate peritonitis, WT MC displayed a clear survival advantage over coengrafted St2(-/-) MC. IL-33 blockade specifically attenuated this survival advantage, confirming IL-33 as the relevant ST2 ligand mediating MC survival in vivo. Together, these data reveal a cell-intrinsic role for the IL-33/ST2 axis in the regulation of apoptosis in MC, identifying thereby a previously unappreciated pathway supporting expansion of the MC population with inflammation.

B cells regulate CD4+ T cell responses to papain following B cell receptor-independent papain uptake.

Papain, a cysteine protease allergen with inherent adjuvant activity, induces potent IL-4 expression by T cells in the popliteal lymph nodes of mice following footpad immunization. In this study, we identify a novel, non-BCR-mediated capacity for B cells to rapidly bind and internalize papain. B cells subsequently regulate the adaptive immune response by enhancing ICOS expression on CD4(+) T cells and amplifying Th2 and follicular helper T cell induction. Ab blockade of ICOS ligand, expressed by popliteal lymph node B cells, but not dendritic cells, at the peak of the response inhibits IL-4 responses in wild-type mice but not B cell-deficient mice. Thus, B cells play a critical role in amplifying adjuvant-dependent Th2 polarization following noncanonical acquisition and internalization of the cysteine protease papain.

Mouse mast cell proteases 4 and 5 mediate epidermal injury through disruption of tight junctions.

We previously established a mast cell (MC)-dependent thermal injury model in mice with ulceration and scar formation that depended on nonredundant functions of mouse MC protease (mMCP)4 and mMCP5. We hypothesized that MC activation is an early event and now find by histology that exocytosis of granule contents occurred by 2 min after thermal injury in wild-type (WT) C57BL/6 mice and in the mMCP4- or mMCP5-deficient mice. The degranulation was equivalent for MCs in the dermis and hypodermis of all three strains, but only the WT mice showed an appreciable increase in epidermal thickness. There was no loss of total MCs, partially degranulated plus intact, during the 4 h of observation. By electron microscopy, MCs in all strains showed early zonal degranulation at 30 s with marked progression in magnitude by 120 s and no mitochondrial injury or cellular necrosis. Concomitantly there was an increase in intercellular spaces indicative of tight junction (TJ) disruption in WT mice but not in the mMCP4- or mMCP5-deficient strains. The desmosomes were intact in all strains. Immunodetection of the TJ protein claudin 4 in WT and mMCP5-deficient mice indicated a significant reduction after scald injury whereas mMCP4(-/-) mice showed no significant changes. Taken together, these findings reveal that a second-degree burn injury can initiate an immediate novel zonal degranulation of MCs throughout all skin layers and a disruption of the epidermal TJs dependent on the nonredundant presence of mMCP4 and mMCP5.

Development of mast cells and importance of their tryptase and chymase serine proteases in inflammation and wound healing.

Mast cells (MCs) are active participants in blood coagulation and innate and acquired immunity. This review focuses on the development of mouse and human MCs, as well as the involvement of their granule serine proteases in inflammation and the connective tissue remodeling that occurs during the different phases of the healing process of wounded skin and other organs. The accumulated data suggest that MCs, their tryptases, and their chymases play important roles in tissue repair. While MCs initially promote healing, they can be detrimental if they are chronically stimulated or if too many MCs become activated at the same time. The possibility that MCs and their granule serine proteases contribute to the formation of keloid and hypertrophic scars makes them potential targets for therapeutic intervention in the repair of damaged skin.

Critical role for mast cell Stat5 activity in skin inflammation.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Here, we show that phospholipase C-ß3 (PLC-ß3)-deficient mice spontaneously develop AD-like skin lesions and more severe allergen-induced dermatitis than wild-type mice. Mast cells were required for both AD models and remarkably increased in the skin of Plcb3(-/-) mice because of the increased Stat5 and reduced SHP-1 activities. Mast cell-specific deletion of Stat5 gene ameliorated allergen-induced dermatitis, whereas that of Shp1 gene encoding Stat5-inactivating SHP-1 exacerbated it. PLC-ß3 regulates the expression of periostin in fibroblasts and TSLP in keratinocytes, two proteins critically involved in AD pathogenesis. Furthermore, polymorphisms in PLCB3, SHP1, STAT5A, and STAT5B genes were associated with human AD. Mast cell expression of PLC-ß3 was inversely correlated with that of phospho-STAT5, and increased mast cells with high levels of phospho-STAT5 were found in lesional skin of some AD patients. Therefore, STAT5 regulatory mechanisms in mast cells are important for AD pathogenesis.

Granule maturation in mast cells: histamine in control.

Mast cells are derived from committed progenitors that originate in the BM. They mature into histochemically distinguishable, metachromatic mast cells containing numerous cytoplasmic secretory granules. Accumulating evidence demonstrates that mast cell granule maturation is very tightly regulated by many factors including different granule components such as proteoglycans. In this issue of the European Journal of Immunology, Nakazawa et al. [Eur. J. Immunol. 2014. 44: 204-214] highlight a role for mast cell derived histamine as another factor critical for mast cell maturation. Using histidine decarboxylase (HDC) deficient mice that are unable to make histamine, they show poorly formed secretory granules and decreased secretory granule protease expression in peritoneal mast cells. Co-culturing BM-derived mast cells with fibroblasts normally drives granule maturation, but HDC-deficient BM-derived mast cells fail to do so. Exogenously provided histamine partly restores granule differentiation as evidenced by increased tryptase and chymase activity, and this is histamine receptor type H4 -dependent. However, H4 -deficient mice have intact granule formation in peritoneal mast cells, suggesting that when HDC is functional, the intrinsic histamine production is sufficient for most granule maturation processes and H4 is dispensable. This study highlights the role of histamine in the regulation of mast cell maturation, although the cytosolic target remains unknown.

Epicutaneous sensitization results in IgE-dependent intestinal mast cell expansion and food-induced anaphylaxis.

BACKGROUND: Sensitization to food antigen can occur through cutaneous exposure. OBJECTIVE: We sought to test the hypothesis that epicutaneous sensitization with food antigen predisposes to IgE-mediated anaphylaxis on oral allergen challenge. METHODS: BALB/c mice were epicutaneously sensitized by repeated application of ovalbumin (OVA) to tape-stripped skin over 7 weeks or orally immunized with OVA and cholera toxin (CT) weekly for 8 weeks and then orally challenged with OVA. Body temperature was monitored, and serum mouse mast cell protease 1 levels were determined after challenge. Tissue mast cell (MC) counts were examined by using chloroacetate esterase staining. Levels of serum OVA-specific IgE and IgG(1) antibodies and cytokines in supernatants of OVA-stimulated splenocytes were measured by means of ELISA. Serum IL-4 levels were measured by using an in vivo cytokine capture assay. RESULTS: Epicutaneously sensitized mice exhibited expansion of connective tissue MCs in the jejunum, increased serum IL-4 levels, and systemic anaphylaxis after oral challenge, as evidenced by decreased body temperature and increased serum mouse mast cell protease 1 levels. Intestinal MC expansion and anaphylaxis were IgE dependent because they did not occur in epicutaneously sensitized IgE(-/-) mice. Mice orally immunized with OVA plus CT did not have increased serum IL-4 levels, expanded intestinal MCs, or anaphylaxis after oral challenge, despite OVA-specific IgE levels and splenocyte cytokine production in response to OVA stimulation, which were comparable with those of epicutaneously sensitized mice. CONCLUSION: Epicutaneously sensitized mice, but not mice orally immunized with antigen plus CT, have expansion of intestinal MCs and IgE-mediated anaphylaxis after single oral antigen challenge. IgE is necessary but not sufficient for food anaphylaxis, and MC expansion in the gut can play an important role in the development of anaphylaxis.