The injured monocyte: The link to chronic critical illness and mortality following injury.

BACKGROUND: This study aimed to understand the altered innate immune response in severely injured patients leading to chronic critical illness (CCI). Specifically, it focused on characterizing the monocyte populations and their correlation with CCI development and long-term complications. METHODS: Over a 3-year period, we monitored patients with severe injuries for up to 1-year postinjury. Chronic critical illness was defined as an ICU stay exceeding 14 days with persistent organ failure. Blood samples were collected on Days 1 and 5 for monocyte phenotypic expression analysis using cytometry by time flight. The monocyte subpopulations studied were classical (CL), intermediate (INT), and nonclassical (NC), along with cell surface receptor expression and activation. RESULTS: Out of 80 enrolled patients, 26 (32.5%) developed CCI. Patients with CCI had more severe injuries (Injury Severity Score, 32.4 + 5.2 vs. 29.6 + 4.1, p = 0.01) and received a higher number of red blood cells (8.9 + 4.1 vs. 4.7 + 3.8 units, p < 0.01) compared with those without CCI. In patients with CCI, the NC monocytes were significantly reduced by over twofold early, and significantly increased later, compared with those without CCI. Moreover, significant changes in intracellular cytokine expression and cell receptors were observed within each monocyte subpopulation in patients with CCI, indicating an increased proinflammatory phenotype but decreased phagocytic capacity and antigen presentation. The development of CCI and the presence of this unique monocyte phenotype were associated with a significantly increased risk of infection, discharge to a long-term care facility, and 1-year mortality of 27%. CONCLUSION: Development of CCI following severe injury is associated with significant long-term morbidity and unacceptably high mortality. The altered NC phenotype with reduced phagocytic capacity and antigen presentation in patients developing CCI after severe injury is appears partially responsible. Early identification of this unique phenotype may help predict and treat patients at risk for CCI, leading to improved outcomes. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

Distinct Epithelial-Innate Immune Cell Transcriptional Circuits Underlie Airway Hyperresponsiveness in Asthma.

Rationale: Indirect airway hyperresponsiveness (AHR) is a highly specific feature of asthma, but the underlying mechanisms responsible for driving indirect AHR remain incompletely understood. Objectives: To identify differences in gene expression in epithelial brushings obtained from individuals with asthma who were characterized for indirect AHR in the form of exercise-induced bronchoconstriction (EIB). Methods: RNA-sequencing analysis was performed on epithelial brushings obtained from individuals with asthma with EIB (n = 11) and without EIB (n = 9). Differentially expressed genes (DEGs) between the groups were correlated with measures of airway physiology, sputum inflammatory markers, and airway wall immunopathology. On the basis of these relationships, we examined the effects of primary airway epithelial cells (AECs) and specific epithelial cell-derived cytokines on both mast cells (MCs) and eosinophils (EOS). Measurements and Main Results: We identified 120 DEGs in individuals with and without EIB. Network analyses suggested critical roles for IL-33-, IL-18-, and IFN-γ-related signaling among these DEGs. IL1RL1 expression was positively correlated with the density of MCs in the epithelial compartment, and IL1RL1, IL18R1, and IFNG were positively correlated with the density of intraepithelial EOS. Subsequent ex vivo modeling demonstrated that AECs promote sustained type 2 (T2) inflammation in MCs and enhance IL-33-induced T2 gene expression. Furthermore, EOS increase the expression of IFNG and IL13 in response to both IL-18 and IL-33 as well as exposure to AECs. Conclusions: Circuits involving epithelial interactions with MCs and EOS are closely associated with indirect AHR. Ex vivo modeling indicates that epithelial-dependent regulation of these innate cells may be critical in indirect AHR and modulating T2 and non-T2 inflammation in asthma.

Siglec-9 is an inhibitory receptor on human mast cells in vitro.

BACKGROUND: Mast cell activation is critical for the development of allergic diseases. Ligation of sialic acid-binding immunoglobin-like lectins (Siglecs), such as Siglec-6, -7, and -8 as well as CD33, have been shown to inhibit mast cell activation. Recent studies showed that human mast cells express Siglec-9, an inhibitory receptor also expressed by neutrophils, monocytes, macrophages, and dendritic cells. OBJECTIVE: We aimed to characterize Siglec-9 expression and function in human mast cells in vitro. METHODS: We assessed the expression of Siglec-9 and Siglec-9 ligands on human mast cell lines and human primary mast cells by real-time quantitative PCR, flow cytometry, and confocal microscopy. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt the SIGLEC9 gene. We evaluated Siglec-9 inhibitory activity on mast cell function by using native Siglec-9 ligands, glycophorin A (GlycA), and high-molecular-weight hyaluronic acid, a monoclonal antibody against Siglec-9, and coengagement of Siglec-9 with the high-affinity receptor for IgE (FcεRI). RESULTS: Human mast cells express Siglec-9 and Siglec-9 ligands. SIGLEC9 gene disruption resulted in increased expression of activation markers at baseline and increased responsiveness to IgE-dependent and IgE-independent stimulation. Pretreatment with GlycA or high-molecular-weight hyaluronic acid followed by IgE-dependent or -independent stimulation had an inhibitory effect on mast cell degranulation. Coengagement of Siglec-9 with FcεRI in human mast cells resulted in reduced degranulation, arachidonic acid production, and chemokine release. CONCLUSIONS: Siglec-9 and its ligands play an important role in limiting human mast cell activation in vitro.

Rhinovirus infection of the airway epithelium enhances mast cell immune responses via epithelial-derived interferons.

BACKGROUND: Mast cells (MCs) within the airway epithelium in asthma are closely related to airway dysfunction, but cross talk between airway epithelial cells (AECs) and MCs in asthma remains incompletely understood. Human rhinovirus (RV) infections are key triggers for asthma progression, and AECs from individuals with asthma may have dysregulated antiviral responses. OBJECTIVE: We utilized primary AECs in an ex vivo coculture model system to examine cross talk between AECs and MCs after epithelial rhinovirus infection. METHODS: Primary AECs were obtained from 11 children with asthma and 10 healthy children, differentiated at air-liquid interface, and cultured in the presence of laboratory of allergic diseases 2 (LAD2) MCs. AECs were infected with rhinovirus serogroup A 16 (RV16) for 48 hours. RNA isolated from both AECs and MCs underwent RNA sequencing. Direct effects of epithelial-derived interferons on LAD2 MCs were examined by real-time quantitative PCR. RESULTS: MCs increased expression of proinflammatory and antiviral genes in AECs. AECs demonstrated a robust antiviral response after RV16 infection that resulted in significant changes in MC gene expression, including upregulation of genes involved in antiviral responses, leukocyte activation, and type 2 inflammation. Subsequent ex vivo modeling demonstrated that IFN-ß induces MC type 2 gene expression. The effects of AEC donor phenotype were small relative to the effects of viral infection and the presence of MCs. CONCLUSIONS: There is significant cross talk between AECs and MCs, which are present in the epithelium in asthma. Epithelial-derived interferons not only play a role in viral suppression but also further alter MC immune responses including specific type 2 genes.

Mast cell-derived factor XIIIA contributes to sexual dimorphic defense against group B streptococcal infections.

Invasive bacterial infections remain a major cause of human morbidity. Group B streptococcus (GBS) are Gram-positive bacteria that cause invasive infections in humans. Here, we show that factor XIIIA-deficient (FXIIIA-deficient) female mice exhibited significantly increased susceptibility to GBS infections. Additionally, female WT mice had increased levels of FXIIIA and were more resistant to GBS infection compared with isogenic male mice. We observed that administration of exogenous FXIIIA to male mice increased host resistance to GBS infection. Conversely, administration of a FXIIIA transglutaminase inhibitor to female mice decreased host resistance to GBS infection. Interestingly, male gonadectomized mice exhibited decreased sensitivity to GBS infection, suggesting a role for gonadal androgens in host susceptibility. FXIIIA promoted GBS entrapment within fibrin clots by crosslinking fibronectin with ScpB, a fibronectin-binding GBS surface protein. Thus, ScpB-deficient GBS exhibited decreased entrapment within fibrin clots in vitro and increased dissemination during systemic infections. Finally, using mice in which FXIIIA expression was depleted in mast cells, we observed that mast cell-derived FXIIIA contributes to host defense against GBS infection. Our studies provide insights into the effects of sexual dimorphism and mast cells on FXIIIA expression and its interactions with GBS adhesins that mediate bacterial dissemination and pathogenesis.

Single-cell analysis identifies the interaction of altered renal tubules with basophils orchestrating kidney fibrosis.

Inflammation is an important component of fibrosis but immune processes that orchestrate kidney fibrosis are not well understood. Here we apply single-cell sequencing to a mouse model of kidney fibrosis. We identify a subset of kidney tubule cells with a profibrotic-inflammatory phenotype characterized by the expression of cytokines and chemokines associated with immune cell recruitment. Receptor-ligand interaction analysis and experimental validation indicate that CXCL1 secreted by profibrotic tubules recruits CXCR2+ basophils. In mice, these basophils are an important source of interleukin-6 and recruitment of the TH17 subset of helper T cells. Genetic deletion or antibody-based depletion of basophils results in reduced renal fibrosis. Human kidney single-cell, bulk gene expression and immunostaining validate a function for basophils in patients with kidney fibrosis. Collectively, these studies identify basophils as contributors to the development of renal fibrosis and suggest that targeting these cells might be a useful clinical strategy to manage chronic kidney disease.

Location of eosinophils in the airway wall is critical for specific features of airway hyperresponsiveness and T2 inflammation in asthma.

BACKGROUND: Eosinophils are implicated as effector cells in asthma, but the functional implications of the precise location of eosinophils in the airway wall is poorly understood. We aimed to quantify eosinophils in the different compartments of the airway wall and associate these findings with clinical features of asthma and markers of airway inflammation. METHODS: In this cross-sectional study, we utilised design-based stereology to accurately partition the numerical density of eosinophils in both the epithelial compartment and the subepithelial space (airway wall area below the basal lamina including the submucosa) in individuals with and without asthma and related these findings to airway hyperresponsiveness (AHR) and features of airway inflammation. RESULTS: Intraepithelial eosinophils were linked to the presence of asthma and endogenous AHR, the type that is most specific for asthma. In contrast, both intraepithelial and subepithelial eosinophils were associated with type 2 (T2) inflammation, with the strongest association between IL5 expression and intraepithelial eosinophils. Eosinophil infiltration of the airway wall was linked to a specific mast cell phenotype that has been described in asthma. We found that interleukin (IL)-33 and IL-5 additively increased cysteinyl leukotriene (CysLT) production by eosinophils and that the CysLT LTC4 along with IL-33 increased IL13 expression in mast cells and altered their protease profile. CONCLUSIONS: We conclude that intraepithelial eosinophils are associated with endogenous AHR and T2 inflammation and may interact with intraepithelial mast cells via CysLTs to regulate airway inflammation.

Mast cell surfaceome characterization reveals CD98 heavy chain is critical for optimal cell function.

BACKGROUND: Mast cells are involved in many distinct pathologic conditions, suggesting that they recognize and respond to various stimuli and thus require a rich repertoire of cell surface proteins. However, mast cell surface proteomes have not been comprehensively characterized. OBJECTIVE: We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease. METHODS: We enriched for glycosylated surface proteins expressed in mouse bone marrow-derived cultured mast cells (BMCMCs) and identified them using mass spectrometry analysis. The presence of novel surface proteins in mast cells was validated by real-time quantitative PCR and flow cytometry analysis in BMCMCs and peritoneal mast cells (PMCs). We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt genes of interest in BMCMCs. RESULTS: The glycoprotein enrichment approach resulted in the identification of 1270 proteins in BMCMCs, 378 of which were localized to the plasma membrane. The most common protein classes among plasma membrane proteins were small GTPases, receptors, and transporters. One such cell surface protein was CD98 heavy chain (CD98hc), encoded by the Slc3a2 gene. Slc3a2 gene disruption resulted in a significant reduction in CD98hc expression, adhesion, and proliferation. CONCLUSIONS: Glycoprotein enrichment coupled with mass spectrometry can be used to identify novel surface molecules in mast cells. Moreover, CD98hc plays an important role in mast cell function.