Highlight of 2023: Advances in mast cells.

In this article for the Highlights of 2023 Series, we consider the growing understanding of mast cell heterogeneity and interactions that has developed from single cell RNA sequencing studies. We also discuss novel concepts concerning mast cell interactions with the central nervous system and evidence for their role in host defense against SARS-CoV-2 infection.

Novel inflammatory mediator profile observed during pediatric heart surgery with cardiopulmonary bypass and continuous ultrafiltration.

BACKGROUND: Cardiopulmonary bypass (CPB) is associated with systemic inflammation, featuring increased levels of circulating pro-inflammatory cytokines. Intra-operative ultrafiltration extracts fluid and inflammatory factors potentially dampening inflammation-related organ dysfunction and enhancing post-operative recovery. This study aimed to define the impact of continuous subzero-balance ultrafiltration (SBUF) on circulating levels of major inflammatory mediators. METHODS: Twenty pediatric patients undergoing cardiac surgery, CPB and SBUF were prospectively enrolled. Blood samples were collected prior to CPB initiation (Pre-CPB Plasma) and immediately before weaning off CPB (End-CPB Plasma). Ultrafiltrate effluent samples were also collected at the End-CPB time-point (End-CPB Effluent). The concentrations of thirty-nine inflammatory factors were assessed and sieving coefficients were calculated. RESULTS: A profound increase in inflammatory cytokines and activated complement products were noted in plasma following CBP. Twenty-two inflammatory mediators were detected in the ultrafiltrate effluent. Novel mediators removed by ultrafiltration included cytokines IL1-Ra, IL-2, IL-12, IL-17A, IL-33, TRAIL, GM-CSF, ET-1, and the chemokines CCL2, CCL3, CCL4, CXCL1, CXCL2 and CXCL10. Mediator extraction by SBUF was significantly associated with molecular mass < 66 kDa (Chi2 statistic = 18.8, Chi2 with Yates' correction = 16.0, p < 0.0001). There was a moderate negative linear correlation between molecular mass and sieving coefficient (Spearman R = - 0.45 and p = 0.02). Notably, the anti-inflammatory cytokine IL-10 was not efficiently extracted by SBUF. CONCLUSIONS: CPB is associated with a burden of circulating inflammatory mediators, and SBUF selectively extracts twenty of these pro-inflammatory factors while preserving the key anti-inflammatory regulator IL-10. Ultrafiltration could potentially function as an immunomodulatory therapy during pediatric cardiac surgery. Trial registration ClinicalTrials.gov, NCT05154864. Registered retrospectively on December 13, 2021. https://clinicaltrials.gov/ct2/show/record/NCT05154864 .

Breastfeeding and the developmental origins of mucosal immunity: how human milk shapes the innate and adaptive mucosal immune systems.

PURPOSE OF REVIEW: Breastfeeding provides passive immunity while the neonatal immune system matures, and may also protect against chronic immune-mediated conditions long after weaning. This review summarizes current knowledge and new discoveries about human milk and mucosal immunity. RECENT FINDINGS: New data suggest that certain microbes in maternal milk may seed and shape the infant gut microbiota, which play a key role in regulating gut barrier integrity and training the developing immune system. Human milk oligosaccharides, best known for their prebiotic functions, have now been shown to directly modulate gene expression in mast and goblet cells in the gastrointestinal tract. Epidemiologic data show a reduced risk of peanut sensitization among infants breastfed by peanut-consuming mothers, suggesting a role for milk-borne food antigens in tolerance development. Cross-fostering experiments in mice suggest the soluble Toll-like receptor 2, found in human milk, may be critical in this process. Finally, interest in human milk antibodies surged during the pandemic with the identification of neutralizing severe acute respiratory syndrome coronavirus 2 antibodies in maternal milk following both natural infection and vaccination. SUMMARY: Human milk provides critical immune protection and stimulation to breastfed infants. Understanding the underlying mechanisms could identify new therapeutic targets and strategies for disease prevention across the lifespan.

Toll-like receptor 2 impacts the development of oral tolerance in mouse pups via a milk-dependent mechanism.

BACKGROUND: The role of breast-feeding in the development of oral tolerance and allergic diseases is controversial, which could be related to variability in milk components. Toll-like receptor 2 (TLR2) is an innate immune receptor implicated in regulating allergic disease development. OBJECTIVES: We examined whether deficiency of maternal TLR2 affects the normal development of oral tolerance and related immune parameters during lactation in a mouse model. METHODS: Heterozygous TLR2+/- pups from wild-type (WT) or TLR2-/- dams were fed either by their biologic dam or a dam of the alternate genotype. Development of oral tolerance to ovalbumin, levels of tolerogenic CD103+ dendritic cells, and regulatory T (Treg) cells, as well as intestinal permeability, were evaluated in these pups. The levels of key immune mediators in milk from TLR2-/- and WT mothers were also examined. RESULTS: Heterozygous TLR2+/- pups that were born to and nursed by TLR2-/- dams exhibited impaired oral tolerance. This was prevented by cross-fostering onto WT (TLR2+/+) dams. Impairments included selective elevation in anti-ovalbumin IgE in plasma following immunization, reduced numbers of tolerogenic dendritic cells and Treg cells in the intestinal tract, and increased intestinal permeability. TLR2 deficiency also affected milk content of insulin-like growth factor-1, IFN-γ, IL-6, and IL-13. CONCLUSION: Our results underline a critical role for TLR2 in regulating milk components that are essential for development of oral tolerance in early life and demonstrate the importance of considering the immune status of nursing mothers in studies of immune development and responses.

Increased mast cell density is associated with decreased fibrosis in human atrial tissue.

Fibrotic remodelling of the atria is poorly understood and can be regulated by myocardial immune cell populations after injury. Mast cells are resident immune sentinel cells present in the heart that respond to tissue damage and have been linked to fibrosis in other settings. The role of cardiac mast cells in fibrotic remodelling in response to human myocardial injury is controversial. In this study, we sought to determine the association between mast cells, atrial fibrosis, and outcomes in a heterogeneous population of cardiac surgical patients, including a substantial proportion of coronary artery bypass grafting patients. Atrial appendage from patients was assessed for collagen and mast cell density by histology and by droplet digital polymerase chain reaction (ddPCR) for mast cell associated transcripts. Clinical variables and outcomes were also followed. Mast cells were detected in human atrial tissue at varying densities. Histological and ddPCR assessment of mast cells in atrial tissue were closely correlated. Patients with high mast cell density had less fibrosis and lower severity of heart failure classification or incidence mortality than patients with low mast cell content. Analysis of a homogeneous population of coronary artery bypass graft patients yielded similar observations. Therefore, evidence from this study suggests that increased atrial mast cell populations are associated with decreased clinical cardiac fibrotic remodelling and improved outcomes, in cardiac surgery patients.

Toll-like receptor 2 activation induces C-C chemokine receptor 2-dependent natural killer cell recruitment to the peritoneum.

Natural killer (NK) cells are innate effector cells with critical roles not only in tumor immunosurveillance and viral immunity, but also in bacterial and fungal infections. Toll-like receptor 2 (TLR2) can be important in the early and sustained immune responses to pathogens and tumors through the induction of cytokines and chemokines that recruit and activate immune effector cells. We investigated the role of TLR2 activation in NK cell recruitment with a view to informing approaches to induce or regulate peritoneal NK cell responses therapeutically. Peritoneal injection of TLR2 activators, including peptidoglycan and the lipopeptides FSL-1 and Pam3 CSK4 , resulted in NK cell recruitment after 16 h with increased NK cell numbers maintained for 48 h. TLR2 activators induced large amounts of CCR2 ligands, but much smaller amounts of CCR5 and CXCR3 ligands. Consistent with this observation, NK cell migration was abrogated in CCR2-deficient mice after peritoneal FSL-1 injection. Adoptive transfer of CCR2-deficient NK cells prior to peritoneal FSL-1 activation confirmed a cell-intrinsic component of CCR2-mediated NK cell migration. TLR2 activation did not induce an activated NK cell phenotype, but significant changes included an increase in the KLRG1+ subset and decreased NKG2D expression. Although not activated in vivo, peritoneal NK cells could be activated by interleukin (IL)-12 and IL-18 ex vivo to express CD69 and interferonγ. These data demonstrate that TLR2-mediated immune activation is a potent inducer of NK cell recruitment via a CCR2-dependent mechanism and that NK cells recruited by this mechanism can respond to additional signals to exert effector cell functions.

Nav1.6 promotes inflammation and neuronal degeneration in a mouse model of multiple sclerosis.

BACKGROUND: In multiple sclerosis (MS) and in the experimental autoimmune encephalomyelitis (EAE) model of MS, the Nav1.6 voltage-gated sodium (Nav) channel isoform has been implicated as a primary contributor to axonal degeneration. Following demyelination Nav1.6, which is normally co-localized with the Na+/Ca2+ exchanger (NCX) at the nodes of Ranvier, associates with ß-APP, a marker of neural injury. The persistent influx of sodium through Nav1.6 is believed to reverse the function of NCX, resulting in an increased influx of damaging Ca2+ ions. However, direct evidence for the role of Nav1.6 in axonal degeneration is lacking. METHODS: In mice floxed for Scn8a, the gene that encodes the α subunit of Nav1.6, subjected to EAE we examined the effect of eliminating Nav1.6 from retinal ganglion cells (RGC) in one eye using an AAV vector harboring Cre and GFP, while using the contralateral either injected with AAV vector harboring GFP alone or non-targeted eye as control. RESULTS: In retinas, the expression of Rbpms, a marker for retinal ganglion cells, was found to be inversely correlated to the expression of Scn8a. Furthermore, the gene expression of the pro-inflammatory cytokines Il6 (IL-6) and Ifng (IFN-γ), and of the reactive gliosis marker Gfap (GFAP) were found to be reduced in targeted retinas. Optic nerves from targeted eyes were shown to have reduced macrophage infiltration and improved axonal health. CONCLUSION: Taken together, our results are consistent with Nav1.6 promoting inflammation and contributing to axonal degeneration following demyelination.

Mast Cell Responses to Viruses and Pathogen Products.

Mast cells are well accepted as important sentinel cells for host defence against selected pathogens. Their location at mucosal surfaces and ability to mobilize multiple aspects of early immune responses makes them critical contributors to effective immunity in several experimental settings. However, the interactions of mast cells with viruses and pathogen products are complex and can have both detrimental and positive impacts. There is substantial evidence for mast cell mobilization and activation of effector cells and mobilization of dendritic cells following viral challenge. These cells are a major and under-appreciated local source of type I and III interferons following viral challenge. However, mast cells have also been implicated in inappropriate inflammatory responses, long term fibrosis, and vascular leakage associated with viral infections. Progress in combating infection and boosting effective immunity requires a better understanding of mast cell responses to viral infection and the pathogen products and receptors we can employ to modify such responses. In this review, we outline some of the key known responses of mast cells to viral infection and their major responses to pathogen products. We have placed an emphasis on data obtained from human mast cells and aim to provide a framework for considering the complex interactions between mast cells and pathogens with a view to exploiting this knowledge therapeutically. Long-lived resident mast cells and their responses to viruses and pathogen products provide excellent opportunities to modify local immune responses that remain to be fully exploited in cancer immunotherapy, vaccination, and treatment of infectious diseases.

Respiratory syncytial virus infection of primary human mast cells induces the selective production of type I interferons, CXCL10, and CCL4.

BACKGROUND: Respiratory syncytial virus (RSV) causes severe respiratory tract infections, which might have a role in the development of airway hyperreactivity. Mast cells are important effector cells in allergy, with sentinel cell roles in host defense. However, the role of mast cells in response to RSV infection is unknown. OBJECTIVE: Human mast cell responses to RSV were investigated with a view to better understanding the role of mast cells in RSV-induced disease. METHODS: Human cord blood-derived mast cells and the HMC-1 mast cell line were exposed to RSV or UV-inactivated RSV. Viral gene and protein expression were evaluated by using PCR and flow cytometry. The expression of interferon-stimulated genes and selected mediators were evaluated by using quantitative PCR and ELISA. RESULTS: Human mast cells expressed multiple RSV genes after exposure to RSV, and a small percentage of mast cells supported RSV antigen protein expression. RSV induced mast cells to upregulate production of chemokines, including CCL4, CCL5, and CXCL10, as well as type I interferons, and interferon-stimulated gene expression. However, production of the granulocyte chemoattractants CXCL8 and CCL11 was not induced. Antibody blockade of the type I interferon receptor on human cord blood-derived mast cells reduced the RSV-mediated induction of CXCL10 and CCL4 but not CCL5. Leukotriene C4 production by mast cells was not enhanced by exposure to RSV. CONCLUSION: Despite low levels of infection, human mast cells produce multiple chemokines in response to RSV through mechanisms that include responses to type I interferons. Such mast cell responses might enhance effector cell recruitment during RSV-induced disease.

Predictors of interleukin-33 and thymic stromal lymphopoietin levels in cord blood.

BACKGROUND: The fetal immune system is a critical window of development. The epithelial cell-derived cytokines, thymic stromal lymphopoietin (TSLP), and interleukin-33 (IL-33) have received attention for their role in allergic responses but not been studied during this critical window. The objectives were to assess correlations among IL-33, TSLP, and IgE in umbilical cord blood samples and identify prenatal predictors of these biomarkers. METHODS: This study utilized data and banked cord blood collected in the Maternal-Infant Research on Environmental Chemicals (MIREC) Study, a trans-Canada cohort study of 2001 pregnant women. Our analytic sample comprised the 1254 women with a singleton, term birth with a cord blood sample. Spearman correlation coefficients (SCC) and logistic regression models were used to examine associations between biomarkers and identify potential predictors of elevated biomarker levels. RESULTS: Thymic stromal lymphopoietin and IL-33 were more strongly correlated with each other (SCC = 0.75, p < 0.0001) than with IgE (IL-33 SCC = 0.14, TSLP SCC = 0.21). Maternal allergy, heavy street traffic, and elevated birth weight were significantly associated with jointly elevated TSLP and IL-33 levels, whereas maternal age and female infant sex were inversely associated with elevated IgE. CONCLUSIONS: In this population of Canadian women and infants, TSLP and IL-33 were detectable in cord blood, more strongly correlated with each other than with IgE, and associated with maternal characteristics indicative of inflammatory responses. This study motivates investigation into the value of cord blood IL-33 and TSLP levels as childhood allergy predictors and raises interesting questions regarding in utero coordinated regulation of these cytokines.

Prenatal exposure to phthalates, bisphenol A and perfluoroalkyl substances and cord blood levels of IgE, TSLP and IL-33.

The fetal time period is a critical window of immune system development and resulting heightened susceptibility to the adverse effects of environmental exposures. Epidemiologists and toxicologists have hypothesized that phthalates, bisphenol A (BPA) and perfluoroalkyl substance have immunotoxic properties. Immunotoxic effects of chemicals may manifest in an altered immune system profile at birth. Immunoglobulin E, thymic stromal lymphopoietin (TSLP), and interleukin-33 (IL-33) are integral in the etiology of childhood allergy and detectable at birth. The objective of this study was to determine the association between maternal levels of phthalates, bisphenol A (BPA), and perfluoroalkyl substances and elevated umbilical cord blood levels of IgE, TSLP, and IL-33. This study utilized data collected in the Maternal-Infant Research on Environmental Chemicals (MIREC) Study, a trans-Canada cohort study of 2001 pregnant women. Of these women, 1258 had a singleton, term birth and cord blood sample. A Bayesian hierarchical model was employed to determine associations between log-transformed continuous variables and immune system biomarkers while adjusting for potential confounding from correlated environmental contaminants. Inverse, nonlinear associations were observed between maternal urinary MCPP levels and elevated levels of both IL-33/TSLP and IgE and between maternal urinary BPA levels and elevated levels of IL-33/TSLP. In this primarily urban Canadian population of pregnant women and their newborns, maternal urinary and plasma concentrations of phthalate metabolites, BPA, and perfluoroalkyl substances were not associated with immunotoxic effects that manifest as increased odds of elevated levels of IgE, TSLP or IL-33.

Maternal exposure to metals and persistent pollutants and cord blood immune system biomarkers.

BACKGROUND: The fetal time period is a critical window of immune system development and resulting heightened susceptibility to the adverse effects of environmental exposures. Epidemiologists and toxicologists have hypothesized that persistent organic pollutants, pesticides and metals have immunotoxic properties. Immunotoxic effects may manifest as an altered immune system profile at birth. Immunoglobulin E, thymic stromal lymphopoietin (TSLP), and interleukin-33 (IL-33) may be implicated in the etiology of childhood allergy and are detectable at birth. The objective of this study was to examine the potential relationship between maternal concentrations of metals, persistent organic pollutants, and pesticides and elevated umbilical cord blood concentrations of IgE, TSLP, and IL-33 in a Canadian birth cohort. METHODS: This study utilized data collected in the Maternal-Infant Research on Environmental Chemicals (MIREC) Study, a trans-Canada cohort study of 2,001 pregnant women. Of these women, 1258 had a singleton, term birth and cord blood sample. Logistic regression was used to determine associations between log-transformed continuous variables and immune system biomarkers. RESULTS: Inverse relationships were observed between lead, DDE, PCB-118, and a summary index of organophosphorous metabolites and jointly elevated concentrations of IL-33 and TSLP. None of the environmental contaminants were associated with increased odds of a high cord blood immune system biomarker concentration. CONCLUSIONS: In this primarily urban Canadian population of pregnant women and their newborns, maternal blood or urine concentrations of persistent organic pollutants, pesticides, and metals were not associated with immunotoxic effects that manifest as increased odds of elevated concentrations of IgE, TSLP or IL-33.

Mast-cell responses to pathogens.

Mast cells have mainly been studied in the setting of allergic disease, but the importance of mast cells for host defence against several pathogens has now been well established. The location of mast cells, which are found closely associated with blood vessels, allows them to have a crucial sentinel role in host defence. The mast cell has a unique 'armamentarium' of receptor systems and mediators for responding to pathogen-associated signals. Studies of this intriguing immune-effector cell provide important insights into the complex mechanisms by which appropriate innate and acquired immune responses are initiated.

Toll-like receptor 2 as a regulator of oral tolerance in the gastrointestinal tract.

Food allergy, other adverse immune responses to foods, inflammatory bowel disease, and eosinophilic esophagitis have become increasingly common in the last 30 years. It has been proposed in the "hygiene hypothesis" that dysregulated immune responses to environmental microbial stimuli may modify the balance between tolerance and sensitization in some patients. Of the pattern recognition receptors that respond to microbial signals, toll-like receptors (TLRs) represent the most investigated group. The relationship between allergy and TLR activation is currently at the frontier of immunology research. Although TLR2 is abundant in the mucosal environment, little is known about the complex relationship between bystander TLR2 activation by the commensal microflora and the processing of oral antigens. This review focuses on recent advances in our understanding of the relationship between TLR2 and oral tolerance, with an emphasis on regulatory T cells, eosinophils, B cells, IgA, intestinal regulation, and commensal microbes.

Ketotifen directly modifies the fibrotic response of human skin fibroblasts.

Fibrosis is a destructive, end-stage disease process. In the skin, it is associated with systemic sclerosis and scarring with considerable health burden. Ketotifen is a clinical antihistamine and mast cell stabilizer. Studies have demonstrated mast cell-dependent anti-fibrotic effects of ketotifen but direct effects on fibroblasts have not been determined. Human dermal fibroblasts were treated with pro-fibrotic transforming growth factor-ß1 (TGFß) followed by ketotifen or control treatments to determine direct effects on fibrotic fibroblasts. Ketotifen impaired TGFß-induced α-smooth muscle actin gene and protein responses and decreased cytoskeletal- and contractility-associated gene responses associated with fibrosis. Ketotifen reduced Yes-associated protein phosphorylation, transcriptional coactivator with PDZ binding motif transcript and protein levels, and phosphorylation of protein kinase B. In a fibroblast-populated collagen gel contraction assay, ketotifen reduced the contractile activity of TGFß-activated fibroblasts. In a murine model of bleomycin-induced skin fibrosis, collagen density and dermal thickness were significantly decreased in ketotifen-treated mice supporting in vitro findings. These results support a novel, direct anti-fibrotic activity of ketotifen, reducing pro-fibrotic phenotypic changes in fibroblasts and reducing collagen fibres in fibrotic mouse skin. Together, these findings suggest novel therapeutic potential and a novel mechanism of action for ketotifen in the context of fibrosis.

Differences in IDO1+ dendritic cells and soluble CTLA-4 are associated with differential clinical responses to methotrexate treatment in rheumatoid arthritis.

Objective: Antigen-presenting dendritic cells (DCs) and monocytes play an essential role in rheumatoid arthritis (RA) pathogenesis, however, their tolerogenic potential remains unclear. Herein, the tolerogenic profiles of DCs are characterized in treatment-naïve RA patients to determine their role to inflammatory arthritis management. Methods: Thirty-six treatment-naïve RA patients were enrolled, of which 62% were non-responders to methotrexate (MTX) monotherapy based on disease activity score (DAS) after 6-months of therapy. DC and monocyte subset frequencies, activation (CD40, CD86, CD209 expression), and tolerogenic profile (intracellular indoleamine-2,3-dioxygenase [IDO1] and cytotoxic T lymphocyte antigen 4 [CTLA-4] expression) were examined in the baseline peripheral blood by multicolor flow-cytometry. Soluble CTLA-4 (sCTLA-4) levels in plasma were measured. Results: DC subsets were decreased in RA compared to healthy controls (HC), and the frequency of conventional DCs (cDC) inversely correlated with inflammatory markers and improvement in disease activity. CD141+ cDC1s were the major IDO1-expressing cells. IDO1+cDC1s were reduced in RA patients compared to HC. The baseline frequency of IDO1+cDC1s inversely correlated with improvement in disease activity. CTLA-4 expression in CD1c+ cDC2s and monocytes was lower in RA patients compared to HC. Moreover, MTX-responders had a significantly lower frequency of IDO1+cDC1 cells and higher level of sCTLA-4 in the plasma compared to MTX non-responders. There was a strong predictive association of low IDO1+cDC1 cells, low sCTLA-4 and non-response to MTX. Conclusions: Our findings reveal altered DC and monocytes immunophenotypes that are associated with RA pathology and treatment response. The frequencies of tolerogenic IDO1+cDC1s and the low level of sCTLA-4 are strongly associated with MTX non-responsiveness and therapeutic outcome. These results suggest that investigation of the association IDO1+cDC1 and sCTLA-4 with response to treatment may be more generalizable to other autoimmune diseases.

AT2R Activation Improves Wound Healing in a Preclinical Mouse Model.

Abnormal skin healing resulting in chronic wounds or hypertrophic scarring remains a major healthcare burden. Here, the antifibrotic angiotensin II type 2 receptor (AT2R) signaling pathway was modulated to determine its impact on cutaneous wound healing. Balb/c mice received two splinted full-thickness wounds. Topical treatments with the selective AT2R agonist compound 21 (C21) and/or selective antagonist PD123319 or saline vehicle were administered until sacrifice on post-wounding days 7 or 10. The rate of wound re-epithelialization was accelerated by PD123319 and combination treatments. In vitro, C21 significantly reduced human fibroblast migration. C21 increased both collagen and vascular densities at days 7 and 10 post-wounding and collagen I:III ratio at day 10, while PD123319 and combination treatments decreased them. Genes associated with regeneration and repair were upregulated by C21, while PD123319 treatment increased the expression of genes associated with inflammation and immune cell chemotaxis. C21 treatment reduced wound total leukocyte and neutrophil staining densities, while PD123319 increased these and macrophage densities. Overall, AT2R activation with C21 yields wounds that mature more quickly with structural, cellular, and gene expression profiles more closely approximating unwounded skin. These findings support AT2R signal modulation as a potential therapeutic target to improve skin quality during wound healing.