HUNK as a key regulator of tumor-associated macrophages in triple negative breast cancer.

Triple-negative breast cancer (TNBC) lacks the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC tumors are not sensitive to endocrine therapy, and standardized TNBC treatment regimens are lacking. TNBC is a more immunogenic subtype of breast cancer, making it more responsive to immunotherapy intervention. Tumor-associated macrophages (TAMs) constitute one of the most abundant immune cell populations in TNBC tumors and contribute to cancer metastasis. This study examines the role of the protein kinase HUNK in tumor immunity. Gene expression analysis using NanoString's nCounter PanCancer Immune Profiling panel identified that targeting HUNK is associated with changes in the IL-4/IL-4 R cytokine signaling pathway. Experimental analysis shows that HUNK kinase activity regulates IL-4 production in mammary tumor cells, and this regulation is dependent on STAT3. In addition, HUNK-dependent regulation of IL-4 secreted from tumor cells induces polarization of macrophages into an M2-like phenotype associated with TAMs. In return, IL-4 induces cancer metastasis and macrophages to produce epidermal growth factor. These findings delineate a paracrine signaling exchange between tumor cells and TAMs regulated by HUNK and dependent on IL-4/IL-4 R. This highlights the potential of HUNK as a target for reducing TNBC metastasis through modulation of the TAM population.

Diminished γδ T Cells during Murine Allergic Skin Inflammation Is Mediated by IL-4 Signaling in Keratinocytes.

Atopic dermatitis results in diminished barrier function and altered production of antimicrobial peptides. Dendritic epidermal T cells (DETCs) play an important role in the wound repair and inflammation process. Our previous work identified an IL-4-dependent loss of DETCs in Stat6VT mice and in the MC903-induced skin inflammation mouse model. However, the mechanisms through which IL-4 mediates the loss of DETCs are unclear. In this study, we show that IL-4Rα germline knockout mice (Il4ra-/-) have increased DETCs, faster wound healing, and increased epidermal differentiation complex gene and fibronectin expression. The absence of IL-4Rα minimized the MC903-induced loss of DETCs, and reciprocal bone marrow chimera experiments in Il4ra-/- and wild-type mice demonstrated structural nonhematopoietic IL-4-responsive cell-mediated DETC homeostasis. Skin keratinocyte-derived IL-15 decreased dramatically in the MC903 model, while injection of IL-15 rescued DETC loss by promoting DETC proliferation and limiting apoptosis. Conditional deletion of IL-4Rα from keratinocytes using Il4rafl/fl K14-Cre mice showed an increase of DETCs, increased IL-15 production, and diminished skin inflammation following wounding. These results suggest that IL-4-dependent effects on DETCs in allergic skin inflammation are mediated by the IL-4Rα receptor of keratinocytes.

Protocol for enrichment and functional analysis of human hematopoietic cells from umbilical cord blood.

Umbilical cord blood (CB) is a donor source for hematopoietic cell therapies. Understanding what drives hematopoietic stem and progenitor cell function is critical to our understanding of the usage of CB in hematopoietic cell therapies. Here, we describe how to isolate and analyze the function of human hematopoietic cells from umbilical CB. This protocol demonstrates assays that measure phenotypic properties and hematopoietic cell potency. For complete details on the use and execution of this protocol, please refer to Broxmeyer et al.1.

TL1A priming induces a multi-cytokine Th9 cell phenotype that promotes robust allergic inflammation in murine models of asthma.

Multi-cytokine-producing Th9 cells secrete IL-9 and type 2 cytokines and mediate mouse and human allergic inflammation. However, the cytokines that promote a multi-cytokine secreting phenotype have not been defined. Tumor necrosis factor superfamily member TL1A signals through its receptor DR3 to increase IL-9. Here we demonstrate that TL1A increases expression of IL-9 and IL-13 co-expressing cells in murine Th9 cell cultures, inducing a multi-cytokine phenotype. Mechanistically, this is linked to histone modifications allowing for increased accessibility at the Il9 and Il13 loci. We further show that TL1A alters the transcription factor network underlying expression of IL-9 and IL-13 in Th9 cells and increases binding of transcription factors to Il9 and Il13 loci. TL1A-priming enhances the pathogenicity of Th9 cells in murine models of allergic airway disease through the increased expression of IL-9 and IL-13. Lastly, in both chronic and memory-recall models of allergic airway disease, blockade of TL1A signaling decreases the multi-cytokine Th9 cell population and attenuates the allergic phenotype. Taken together, these data demonstrate that TL1A promotes the development of multi-cytokine Th9 cells that drive allergic airway diseases and that targeting pathogenic T helper cell-promoting cytokines could be an effective approach for modifying disease.

Access to routine health care and awareness of hypertension status among adults: Results from the National Health and Nutrition Examination Survey, 2011-2018.

INTRODUCTION: Hypertension is a growing pandemic affecting over 1 billion people worldwide; about 46% of people with hypertension are unaware. METHOD: Data from the National Health and Nutrition Examination Survey (NHANES) 2011-2018 were analyzed to assess the relationship between access to a routine place of care and undiagnosed hypertension in adults aged 18 to 64 years old. We defined undiagnosed hypertension as those meeting the 2017 American Heart Association's guidelines for stage 1 or 2 hypertension who reported not being told by their healthcare provider that they had hypertension. We used a multivariable Poisson regression model to assess the relationship between access to a routine place of care and undiagnosed hypertension. RESULT: The final analytic sample was 5345 hypertensive American adults, with 56% unaware of their status. The results indicate that lack of awareness of hypertension status was highest among those without a routine place of care [PR = 1.20, CI = (1.12-1.29), p < 0.001] compared to those with access to a routine place of care, after adjustment for sociodemographic and clinical characteristics. CONCLUSION: Access to a routine place of care in a non-emergency department setting is essential to reduce the rate of undiagnosed hypertension among American adults. Policymakers should implement policies to address the shortage of primary care providers and increase access to a routine place of care.

Relationship Between Alcohol Use and Firearm-Involved Suicide: Findings From the National Violent Death Reporting System, 2003-2020.

INTRODUCTION: Acute alcohol intoxication is a contributing factor in firearm-involved suicides. However, knowledge of the relationship between alcohol intoxication and firearm-involved suicide by age and sex (defined herein as the biological sex of the decedent) is limited. The purpose of the current study was to evaluate the sex- and age group-specific relationship between alcohol intoxication and firearm-involved suicide. METHODS: Data from the National Violent Death Reporting System, 2003-2020, on suicide decedents (18+ years of age) were utilized. Age-group- and sex-specific multivariate binary logistic regression analyses were conducted. Statistical analyses were performed in 2023. RESULTS: Alcohol intoxication (i.e., having a blood alcohol concentration of 0.08 g/dL or more) was significantly associated with using a firearm as the method of suicide for young (18-34 years; relative risk (RR)=1.31, 95% CI: 1.22-1.40) and middle-aged (35-64 years; RR=1.34, 95% CI: 1.27-1.39) females but not among older females (65+ years; RR=1.01, 95% CI: 0.87-1.17). Among males, the association was significant for all age-groups (young: RR=1.28, 95% CI: 1.25-1.30; middle-aged: RR=1.17, 95% CI: 1.15-1.19; and older: RR=1.04, 95% CI: 1.01-1.07). CONCLUSIONS: Among males of all ages and young and middle-aged females, alcohol intoxication was associated with increased risk of suicide by firearm-an extremely lethal method that accounts for a majority of suicides in the U.S.-compared to their non-intoxicated counterparts. Interventions targeting excessive alcohol consumption may be effective in reducing suicide mortality rates.

Biofilm-derived oxylipin 10-HOME-mediated immune response in women with breast implants.

This study investigates a mechanistic link of bacterial biofilm-mediated host-pathogen interaction leading to immunological complications associated with breast implant illness (BII). Over 10 million women worldwide have breast implants. In recent years, women have described a constellation of immunological symptoms believed to be related to their breast implants. We report that periprosthetic breast tissue of participants with symptoms associated with BII had increased abundance of biofilm and biofilm-derived oxylipin 10-HOME compared with participants with implants who are without symptoms (non-BII) and participants without implants. S. epidermidis biofilm was observed to be higher in the BII group compared with the non-BII group and the normal tissue group. Oxylipin 10-HOME was found to be immunogenically capable of polarizing naive CD4+ T cells with a resulting Th1 subtype in vitro and in vivo. Consistently, an abundance of CD4+Th1 subtype was observed in the periprosthetic breast tissue and blood of people in the BII group. Mice injected with 10-HOME also had increased Th1 subtype in their blood, akin to patients with BII, and demonstrated fatigue-like symptoms. The identification of an oxylipin-mediated mechanism of immune activation induced by local bacterial biofilm provides insight into the possible pathogenesis of the implant-associated immune symptoms of BII.

STAT3 Signaling Heterogeneity Underlies Cytokine-Expressing Fate in Th17 Cultures.

The polarization of naive Th cells into differentiated subsets in vitro was a powerful approach to define the development and function of Th cells in vivo. Th cell cultures identified cytokines that promote polarization and defined the phenotype and stability of differentiated cells. One of the limitations of this approach is the heterogeneity of the differentiated culture, essentially with regard to what proportion of the culture is secreting the hallmark cytokine of interest. This heterogeneity has always been puzzling because all cells in the culture have been exposed to identical culture conditions. We examined this phenomenon using an Il17f lineage-tracing allele (Cost, Cre on seventeen transcript) crossed to stop-flox Rosa-YFP (yellow fluorescent protein) mice. We found that less than half of the cells in a Th17 culture become lineage-positive during a differentiation culture and that it is primarily cells that are lineage-positive that produce cytokines when cultures are restimulated after differentiation. We sorted and analyzed YFP-positive and YFP-negative cells and found similar expression of many Th17 transcription factors, although YFP-negative cells had increased expression of other lineage-defining transcription factors. We observed that YFP-negative cells had diminished expression of Stat3 and Il6ra, as well as decreased STAT3 activation. YFP-negative cells transduced with active STAT3 had significant increases in IL-17A expression, without increases in Th17 transcription factors. Taken together, these data suggest that there is a threshold of STAT3 activation that is required for efficient Th17 differentiation, and that even in a culture of homogeneous naive T cells there is heterogeneity in the receipt of early cytokine signals.

Insights into highly engraftable hematopoietic cells from 27-year cryopreserved umbilical cord blood.

Umbilical cord blood transplantation is a life-saving treatment for malignant and non-malignant hematologic disorders. It remains unclear how long cryopreserved units remain functional, and the length of cryopreservation is often used as a criterion to exclude older units. We demonstrate that long-term cryopreserved cord blood retains similar numbers of hematopoietic stem and progenitor cells compared with fresh and recently cryopreserved cord blood units. Long-term cryopreserved units contain highly functional cells, yielding robust engraftment in mouse transplantation models. We also leverage differences between units to examine gene programs associated with better engraftment. Transcriptomic analyses reveal that gene programs associated with lineage determination and oxidative stress are enriched in high engrafting cord blood, revealing potential molecular markers to be used as potency markers for cord blood unit selection regardless of length of cryopreservation. In summary, cord blood units cryopreserved for extended periods retain engrafting potential and can potentially be used for patient treatment.

Combination of Lactate and Base Deficit Levels at Admission to Predict Mortality in Blunt Trauma Patients.

INTRODUCTION: Elevated lactate levels are associated with increased mortality in both trauma and non-trauma patients. The relation between base deficit (BD) and mortality is less clear. Traumatologists debate the utility of elevated lactate (EL) versus BD in predicting mortality. We hypothesized that EL (2mmol/L to 5mmol/L) and BD (≤-2mmol/L) in combination could predict mortality in blunt trauma patients.  Methods: This is a retrospective analysis of the trauma registry from 2012 to 2021 at a level 1 trauma center. Blunt trauma patients with admission lactate and BD values were included in the analysis. Exclusion criteria were age <18, penetrating trauma, unknown mortality, and unknown lactate or BD. Logistics regression of the total 5153 charts showed 93% of the patients presented with lactate levels <5mmol/L, therefore patients with lactate >5mmol/L were excluded as outliers. The primary outcome was mortality. RESULTS: A total of 4794 patients (151 non-survivors) were included in the analysis. Non-survivors had higher rates of EL + BD (35.8% vs. 14.4%, p <0.001). When comparing survivors and non-survivors, EL + BD (OR 5.69), age >65 (5.17), injury severity score (ISS) >25 (8.87), Glasgow coma scale <8 (8.51), systolic blood pressure (SBP) <90 (4.2), and ICU admission (2.61) were significant predictors of mortality. Other than GCS <8 and ISS >25, EL + BD had the highest odds of predicting mortality. CONCLUSION: Elevated lactate + BD on admission in combination represents a 5.6-fold increase in mortality in blunt trauma patients and can be used to predict a patient's outcome on admission. This combination variable provides an additional early data point to identify patients at elevated risk of mortality at the moment of admission.

The World according to IL-9.

Among the cytokines regulating immune cells, IL-9 has gained considerable attention for its ability to act on multiple cell types as a regulator of beneficial and pathologic immune responses. Yet, it is still not clearly defined how IL-9 impacts immune responses. IL-9 demonstrates a remarkable degree of tissue-specific functionality and has cellular sources that vary by tissue site and the context of the inflammatory milieu. Here, we provide perspective to summarize the biological activities of IL-9 and highlight cell type-specific roles in the immune pathogenesis of diseases. This perspective will be important in defining the diseases where targeting IL-9 as a therapeutic strategy would be beneficial and where it has the potential to complicate clinical outcomes.

Loricrin and Cytokeratin Disorganisation in Severe Forms of Periodontitis.

OBJECTIVE: The aim of this research was to investigate the role of the cornified epithelium, the outermost layer of the oral mucosa, engineered to prevent water loss and microorganism invasion, in severe forms of periodontitis (stage III or IV, grade C). METHODS: Porphyromonas gingivalis, a major periodontal disease pathogen, can affect cornified epithelial protein expression through chronic activation of signal transducer and activator of transcription 6 (Stat6). We used a mouse model, Stat6VT, that mimics this to determine the effects of barrier defect on P gingivalis-induced inflammation, bone loss, and cornified epithelial protein expression, and compared histologic and immunohistologic findings with tissues obtained from human controls and patients with stage III and IV, grade C disease. Alveolar bone loss in mice was assessed using micro-computerised tomography, and soft tissue morphology was qualitatively and semi-quantitatively assessed by histologic examination for several proteins, including loricrin, filaggrin, cytokeratin 1, cytokeratin 14, a proliferation marker, a pan-leukocyte marker, as well as morphologic signs of inflammation. Relative cytokine levels were measured in mouse plasma by cytokine array. RESULTS: In the tissues from patients with periodontal disease, there were greater signs of inflammation (rete pegs, clear cells, inflammatory infiltrates) and a decrease and broadening of expression of loricrin and cytokeratin 1. Cytokeratin 14 expression was also broader and decreased in stage IV. P gingivalis-infected Stat6VT mice showed greater alveolar bone loss in 9 out of 16 examined sites, and similar patterns of disruption to human patients in expression of loricrin and cytokeratins 1 and 14. There were also increased numbers of leukocytes, decreased proliferation, and greater signs of inflammation compared with P gingivalis-infected control mice. CONCLUSIONS: Our study provides evidence that changes in epithelial organisation can exacerbate the effects of P gingivalis infection, with similarities to the most severe forms of human periodontitis.

The Dichotomy of Interleukin-9 Function in the Tumor Microenvironment.

Interleukin 9 (IL-9) is a cytokine with potent proinflammatory properties that plays a central role in pathologies such as allergic asthma, immunity to parasitic infection, and autoimmunity. More recently, IL-9 has garnered considerable attention in tumor immunity. Historically, IL-9 has been associated with a protumor function in hematological malignancies and an antitumor function in solid malignancies. However, recent discoveries of the dynamic role of IL-9 in cancer progression suggest that IL-9 can act as both a pro- or antitumor factor in various hematological and solid malignancies. This review summarizes IL-9-dependent control of tumor growth, regulation, and therapeutic applicability of IL-9 blockade and IL-9-producing cells in cancer.

Dendritic Cells as a Nexus for the Development of Multiple Sclerosis and Models of Disease.

Multiple sclerosis (MS) results from an autoimmune attack on the central nervous system (CNS). Dysregulated immune cells invade the CNS, causing demyelination, neuronal and axonal damage, and subsequent neurological disorders. Although antigen-specific T cells mediate the immunopathology of MS, innate myeloid cells have essential contributions to CNS tissue damage. Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that promote inflammation and modulate adaptive immune responses. This review focuses on DCs as critical components of CNS inflammation. Here, evidence from studies is summarized with animal models of MS and MS patients that support the critical role of DCs in orchestrating CNS inflammation.

Factor VIII trafficking to CD4+ T cells shapes its immunogenicity and requires several types of antigen-presenting cells.

Despite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in ∼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.