Functional and Phenotypic Characterization of Siglec-6 on Human Mast Cells

Identification of Biological and Pharmaceutical Mast Cell‐ and Basophil‐Related Targets

A major problem in SARS-CoV-2-infected patients is the massive tissue inflammation in certain target organs, including the lungs. Mast cells (MC), basophils (BA), and eosinophils (EO) are key effector cells in inflammatory processes. These cells have recently been implicated in the pathogenesis of SARS-CoV-2 infections. We explored coronavirus receptor (CoV-R) expression profiles in primary human MC, BA, and EO, and in related cell lines (HMC-1, ROSA, MCPV-1, KU812, and EOL-1). As determined using flow cytometry, primary MC, BA, and EO, and their corresponding cell lines, displayed the CoV-R CD13 and CD147. Primary skin MC and BA, as well as EOL-1 cells, also displayed CD26, whereas primary EO and the MC and BA cell lines failed to express CD26. As assessed using qPCR, most cell lines expressed transcripts for CD13, CD147, and ABL2, whereas ACE2 mRNA was not detectable, and CD26 mRNA was only identified in EOL-1 cells. We also screened for drug effects on CoV-R expression. However, dexamethasone, vitamin D, and hydroxychloroquine did not exert substantial effects on the expression of CD13, CD26, or CD147 in the cells. Together, MC, BA, and EO express distinct CoV-R profiles. Whether these receptors mediate virus-cell interactions and thereby virus-induced inflammation remains unknown at present.

Control of Pseudomonas aeruginosa Skin Infections in Mice Is Mast Cell-Dependent

In mast cells, endo- and exocytotic pathways are central to the (patho)physiological release of pro-inflammatory mediators, linking intracellular signaling with immune communication. Proton-activated chloride (PAC) channel mediates acid-sensitive outwardly rectifying anion channel (ASOR/PAORAC) currents, however, its physiological functions are poorly understood.Using electrophysiology, live-cell imaging, electron microscopy, and functional assays, we investigate the role of PAC in human mast cells.We detected ASOR/PAORAC in primary human mast cells and mast cell lines and demonstrated its essential role in vesicular signaling. PAC knockout reduced vesicular pH, increased endocytosis, decreased exocytosis, and disrupted endolysosomal homeostasis. Upon activation of exocytosis, the lack of PAC reduced CD107a (LAMP-1) surface expression. PAC-deficient cells also displayed increased colocalization of lysosomes and mitochondria, elevated ROS levels, and the appearance of C-shaped mitochondria, suggesting that PAC regulates inter-organelle stress signaling. Functionally, PAC knockout impaired mitochondrial respiration, linking ion channel activity to mast cell metabolic adaptation.These findings establish PAC as a key regulator of endo- and exocytosis-dependent signaling and lysosomal-mitochondrial stress response in human mast cells, highlighting its physiological relevance and potential as a therapeutic target in mast cell-associated disorders.

Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells. Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method. IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells. For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.

Mast cells in the liver.

Identification of Novel Mast Cell Activators Using Cell-Based High-Throughput Screening

BackgroundThe KIT receptor tyrosine kinase and its ligand, stem cell factor (SCF) control proliferation and survival of mast cells. Thus, targeting KIT signaling may show promise for the treatment of allergic diseases involving mast cells. Recently, we discovered a new compound MOD000001 as a potential small-molecule KIT kinase inhibitor by using an in silico approach. ObjectiveThis study determined whether MOD000001 is highly-selective to KIT, inhibits KIT signaling in mast cells, and affects IgE-mediated mast cell activation. MethodsThe interaction of MOD000001 with 468 human kinases and its inhibitory activity against KIT were profiled and evaluated by using KinomeScan and cell-free kinase assays, respectively. The effects of MOD000001 on SCF-dependent signaling were examined by using primary mouse and human mast cells. The effects of MOD000001 on SCF-induced degranulation and passive cutaneous anaphylactic (PCA) reaction were examined in mice. ResultsMOD000001 interacted with KIT and inhibited KIT kinase activity with high selectivity. MOD000001 suppressed SCF-induced KIT signaling in mouse and human mast cells and in mice. PCA reaction was suppressed in mice treated with MOD000001 both for a short-term (1 week) and a long-term (7 weeks). Mice treated with MOD000001 for a long-term, but not for a short-term, showed skin mast cell reduction. ConclusionsMOD000001 is a highly selective KIT inhibitor that can suppress IgE-mediated mast cell activation in vivo. MOD000001 may do so by reducing tissue mast cell numbers or by other unknown mechanisms. The findings suggest potential benefits of MOD000001 for allergic diseases involving IgE-mediated mast cell activation.

CD4+ T cell infiltration is known to occur in tissues at sites of mast cell activation. The molecules produced and released by mast cells that account for this lymphocyte accumulation are poorly characterized. Here we report that a CD4+ T cell chemoattractant cytokine, IL-16, is stored preformed in bone marrow-cultured human mast cells and a human mast cell line, HMC-1, as demonstrated by intracytoplasmic cytokine staining and flow cytometry, and in human lung mast cells, as detected by immunohistochemistry. In response to the anaphylatoxin, C5a, or to PMA treatment, IL-16 mRNA transcripts detected by Northern blot analysis in HMC-1 cells increased 6- to 10-fold. HMC-1 cell lysates and activated supernatants contained IL-16 protein, as demonstrated by both ELISA and in vitro lymphocyte chemotaxis assays, the latter of which was blocked 59 to 88% by the addition of neutralizing Ab to recombinant human IL-16. IL-16 bioactivity was detected in the supernatants 2 to 4 h after PMA or C5a activation, and this activity remained elevated through 24 h. The capacity of human mast cells to synthesize and release biologically active IL-16 provides a possible link between mast cell activation and the accumulation of T cells in mast cell-dependent inflammation, thus amplifying the immune response and perpetuating the pathologic process.

Mast cells (MC) are present in most vascularized tissues around the vasculature likely exerting immunomodulatory functions. Endowed with diverse mediators, resident MC represent first-line fine-tuners of local microenvironment. Sphingosine-1-phosphate (S1P) functions as a pluripotent signaling sphingolipid metabolite in health and disease. S1P formation occurs at low levels in resting MC and is upregulated upon activation. Its export can result in type 2 S1P receptor- (S1PR2-) mediated stimulation of MC, further fueling inflammation. However, the role of S1PR2 ligation in proangiogenic vascular endothelial growth factor- (VEGF-) A and matrix metalloproteinase- (MMP-) 2 release from MC is unknown. Using a preclinical MC-dependent model of acute allergic responses and in vitro stimulated primary mouse bone marrow-derived MC (BMMC) or human primary skin MC, we report that S1P signaling resulted in substantial amount of VEGF-A release. Similar experiments using S1pr2-deficient mice or BMMC or selective S1P receptor agonists or antagonists demonstrated that S1P/S1PR2 ligation on MC is important for VEGF-A secretion. Further, we show that S1P stimulation triggered transcriptional upregulation of VEGF-A and MMP-2 mRNA in human but not in mouse MC. S1P exposure also triggered MMP-2 secretion from human MC. These studies identify a novel proangiogenic axis encompassing MC/S1P/S1PR2 likely relevant to inflammation.

Mast cell and basophil activation contributes to inflammation, bronchoconstriction, and airway hyperresponsiveness in asthma. Because IL-33 expression is inflammation inducible, we investigated IL-33-mediated effects in concert with both IgE-mediated and IgE-independent stimulation. Because the HMC-1 mast cell line can be activated by GPCR and RTK signaling, we studied the effects of IL-33 on these pathways. The IL-33- and SCF-stimulated HMC-1 cells were co-cultured with human lung fibroblasts and airway smooth muscle cells in a collagen gel contraction assay. IL-33 effects on IgE-mediated activation were studied in primary mast cells and basophils. IL-33 synergized with adenosine, C5a, SCF, and NGF receptor activation. IL-33-stimulated and SCF-stimulated HMC-1 cells demonstrated enhanced collagen gel contraction when cultured with fibroblasts or smooth muscle cells. IL-33 also synergized with IgE receptor activation of primary human mast cells and basophils. IL-33 amplifies inflammation in both IgE-independent and IgE-dependent responses.

Vitamin D and Atopy

Mast cells play an important role in both allergy and innate immunity. Recently, we demonstrated an active interaction between human mast cells and Pseudomonas aeruginosa leading to the production of multiple cytokines. Here, we show that both primary cultured human cord blood-derived mast cells and the human mast cell line HMC-1 undergo apoptosis as determined by single-stranded DNA (ssDNA) formation after stimulation with P. aeruginosa exotoxin A (ETA), a major toxin produced by this bacterium. ETA-induced ssDNA formation was completely inhibited by Z-VAD (where Z is benzyloxycarbonyl), which blocks multiple caspases, suggesting a role for caspases in this process. Active caspase-3 formation in mast cells after an ETA challenge was detected by both Western blotting and flow cytometry analysis. ETA-induced caspase-3 activity in human mast cells was demonstrated by the detection of a characteristic 23 kDa product of D4-GDI (where GDI is guanine nucleotide dissociation inhibitor), an endogenous caspase-3 substrate. Interestingly, a specific caspase-8 inhibitor, Z-IETD-fmk (where fmk is fluoromethyl ketone), blocked ETA-induced cleavage of D4-GDI, but a caspase-9 inhibitor (Z-LEHD-fmk) did not. Treatment of mast cells with caspase-3 inhibitor Z-DEVD-fmk or caspase-8 inhibitor Z-IETD-fmk reduced the generation of ssDNA induced by ETA, suggesting a role for caspase-8 and -3 in ETA-induced mast cell apoptosis. Furthermore, treatment of mast cells with ETA induced decreases of the short form and a long form (p43) of Fas-associated death domain protein (FADD)-like interleukin-1beta-converting enzyme (FLICE) (caspase-8)-inhibitory proteins (FLIPs), which are endogenous caspase-8 inhibitors. Taken together, these results suggest that ETA-induced mast cell apoptosis involves down-regulation of antiapoptotic proteins, FLIPs, and activation of caspase-8 and -3 pathways.

Mast cells are tissue-resident immune cells widely present in various tissues, such as the gastrointestinal lining, skin, and respiratory epithelium. Their ability to store and rapidly release potent mediators, including inflammatory cytokines and proteases, within minutes of activation makes mast cells a crucial first line of defence in protecting the host against threats and regulating pathological conditions such as food allergies. During food allergy, mast cells release a variety of inflammatory mediators responsible for the rapid onset of gastrointestinal symptoms, including diarrhoea, abdominal pain, and vomiting. Currently, there is no cure for food allergies, and existing treatments focus primarily on symptom management, often with side effects like drowsiness, nausea, and headaches. Consequently, allergen avoidance remains the most effective strategy, but it can lead to nutritional deficiencies, particularly in children allergic to milk and eggs. Thus, alternative and more effective treatments are urgently needed. Kappa opioid receptors (KORs) are widely expressed throughout the human body, including in the brain, spinal cord, gastrointestinal tract, and on various immune cells. Previous studies have shown that KOR modulation using KOR-stimulating compounds significantly improved allergic diarrhoea in a mouse model, and this beneficial effect was prevented when KOR was blocked. These findings highlight the potential role of KOR in food allergy. Although the underlying mechanisms of KOR modulation in food allergy remain unclear, it is hypothesised that KOR activation may alleviate allergic reactions by regulating immune cells, particularly mast cells, and by directly affecting gastrointestinal processes such as motility and fluid absorption. In this thesis, I investigated the effects of KOR modulation using high-affinity KOR agonists, such as U50,488H and nalfurafine, in both in vitro and in vivo models. I optimised a high-purity and functional protocol for generating bone marrow-derived mast cells (BMMCs) to explore the direct effects of KOR agonists on mast cell activation in vitro. All tested KOR agonists, including nalfurafine and U50,488H, successfully inhibited mast cell activation to levels comparable to the positive control, clemastine fumarate. Notably, nalfurafine emerged as the most potent agonist, effectively inhibiting mast cell activation at a concentration as low as 0.5 nM, while U50,488H required a much higher concentration of 1000 nM to achieve similar effects. Interestingly, both agonists exhibited an inverted bell-shaped dose-response curve, with high efficacy at lower concentrations but diminished effects at higher concentrations. Moreover, the inhibitory effects of KOR agonists on mast cell activation were abolished in KOR-deficient BMMCs, confirming that the effects were mediated through the KOR pathway and validating the specificity of the agonists. In addition to the in vitro experiments, I developed a food allergy model to evaluate the in vivo effects of nalfurafine and U50,488H. Both agonists significantly improved clinical symptoms during disease onset, demonstrating their potential as therapeutic agents for managing food allergy symptoms. However, when nalfurafine was administered prophylactically before immunisation, there was no significant effect on the manifestation of food allergies. This suggests that while nalfurafine is effective at alleviating symptoms once the disease is established, it does not prevent the initial sensitisation process that drives food allergies. This thesis also highlights the impact of genetic differences between inbred mouse strains C57BL/6 and BALB/cByJ in both BMMC cultures and the food allergy model. BMMCs from the BALB/cByJ strain were less efficient in culture generation compared to those from the C57BL/6 strain. Conversely, in the food allergy model, C57BL/6 mice exhibited milder food allergy symptoms compared to BALB/cByJ mice. These findings underscore the importance of considering genetic background when designing and interpreting immunological experiments. Overall, the findings presented in this thesis provide valuable insights into the role of KOR in modulating immune responses, particularly in the context of mast cell activation and food allergy. Furthermore, the study emphasises the influence of genetic background on experimental outcomes, with notable differences between C57BL/6 and BALB/cByJ mice in both BMMC culture efficiency and the severity of food allergy symptoms. These findings advance our understanding of KOR modulation in immune responses and suggest potential avenues for developing targeted therapies to manage food allergy and other mast cell-related conditions.

A broad variety of microbes are present in atherosclerotic plaques and chronic bacterial infection increases the risk of atherosclerosis by mechanisms that have remained vague. One possible mechanism is that bacteria or bacterial products activate plaque mast cells that are known to participate in the pathogenesis of atherosclerosis. Here, we show by real-time PCR analysis and ELISA that Chlamydia pneumoniae (Cpn) and a periodontal pathogen, Aggregatibacter actinomycetemcomitans (Aa), both induce a time and concentration-dependent expression and secretion of interleukin 8 (IL-8), tumour necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) by cultured human peripheral blood-derived mast cells, but not anti-inflammatory molecules, such as IL-10 or transforming growth factor β1 (TGF-β1). The IL-8 and MCP-1 responses were immediate, whereas the onset of TNF-α secretion was delayed. The Cpn-mediated pro-inflammatory effect was attenuated when the bacteria were inactivated by UV-treatment. Human monocyte-derived macrophages that were pre-infected with Cpn also induced a significant pro-inflammatory response in human mast cells, both in cocultures and when preconditioned media from Cpn-infected macrophages were used. Intranasal and intravenous administration of live Cpn and Aa, respectively induced an accumulation of activated mast cells in the aortic sinus of apolipoprotein E-deficient mice, however, with varying responses in the systemic levels of lipopolysaccharide (LPS) and TNF-α. Pro-atherogenic Cpn and Aa induce a pro-inflammatory response in cultured human connective tissue-type mast cells and activation of mouse aortic mast cells in vivo.