Systemic neutrophil degranulation and emergency granulopoiesis in patients with Clostridioides difficile infection.

OBJECTIVES: Clostridioides difficile infection (CDI) is characterized by neutrophilia in blood, with a high leukocyte count accompanying severe infection. In this study, we characterized peripheral blood neutrophil activation and maturity in CDI by (i) developing a method to phenotype stored neutrophils for disease-related developmental alterations and (ii) assessing neutrophil-associated biomarkers. METHODS: We stored fixed leukocytes from blood collected within 24 h of diagnosis from a cohort of hospitalized patients with acute CDI. Additional study cohorts included recurrent CDI patients at time of and two months after FMT therapy and a control healthy cohort. We assessed levels of neutrophil surface markers CD66b, CD11b, CD16 and CD10 by flow cytometry. Plasma neutrophil elastase and lipocalin-2 were measured using ELISA, while G-CSF, GM-CSF and cytokines were measured using O-link Proteomic technology. RESULTS: CD66b+ neutrophil abundance assessed by flow cytometry correlated well with complete blood counts, establishing that neutrophils in stored blood are sufficiently well-preserved for phenotyping by flow cytometry. Neutrophil abundance was significantly increased in CDI patients compared to healthy controls. Emergency granulopoiesis in acute CDI patients was evidenced by lower neutrophil surface expression of CD10, CD11b and CD16. CD10+ staining of neutrophils started to recover within 3-7 days of CDI treatment. Neutrophil activation and degranulation were higher in acute CDI as assessed by plasma neutrophil elastase and lipocalin-2. Biomarker levels in immunocompetent subjects were associated with recurrence and fatal outcomes. CONCLUSIONS: Neutrophil activation and emergency granulopoiesis characterize the early immune response in acute CDI, with plasma degranulation biomarkers predictive of disease severity.

The impact of existing total anti-toxin B IgG immunity in outcomes of recurrent Clostridioides difficile infection.

Late anti-toxin-B humoral immunity acquired after treatment is important for preventing recurrent Clostridioides difficile infection. We prospectively-measured anti-toxin-B IgG and neutralization titers at diagnosis as potential early predictors of recurrence. High anti-toxin-B-IgG/neutralizing antibodies were associated with short-lasting protection within 6-weeks, however, no difference in recurrence risk was observed by 90-days post-infection.

Novel Biomarkers, Including tcdB PCR Cycle Threshold, for Predicting Recurrent Clostridioides difficile Infection.

Traditional clinical models for predicting recurrent Clostridioides difficile infection do not perform well, likely owing to the complex host-pathogen interactions involved. Accurate risk stratification using novel biomarkers could help prevent recurrence by improving underutilization of effective therapies (i.e., fecal transplant, fidaxomicin, bezlotoxumab). We used a biorepository of 257 hospitalized patients with 24 features collected at diagnosis, including 17 plasma cytokines, total/neutralizing anti-toxin B IgG, stool toxins, and PCR cycle threshold (CT) (a proxy for stool organism burden). The best set of predictors for recurrent infection was selected by Bayesian model averaging for inclusion in a final Bayesian logistic regression model. We then used a large PCR-only data set to confirm the finding that PCR CT predicts recurrence-free survival using Cox proportional hazards regression. The top model-averaged features were (probabilities of >0.05, greatest to least): interleukin 6 (IL-6), PCR CT, endothelial growth factor, IL-8, eotaxin, IL-10, hepatocyte growth factor, and IL-4. The accuracy of the final model was 0.88. Among 1,660 cases with PCR-only data, cycle threshold was significantly associated with recurrence-free survival (hazard ratio, 0.95; P < 0.005). Certain biomarkers associated with C. difficile infection severity were especially important for predicting recurrence; PCR CT and markers of type 2 immunity (endothelial growth factor [EGF], eotaxin) emerged as positive predictors of recurrence, while type 17 immune markers (IL-6, IL-8) were negative predictors. In addition to novel serum biomarkers (particularly, IL-6, EGF, and IL-8), the readily available PCR CT may be critical to augment underperforming clinical models for C. difficile recurrence.

Cartilage-Specific Knockout of the Mechanosensory Ion Channel TRPV4 Decreases Age-Related Osteoarthritis.

Osteoarthritis (OA) is a progressive degenerative disease of articular cartilage and surrounding tissues, and is associated with both advanced age and joint injury. Biomechanical factors play a critical role in the onset and progression of OA, yet the mechanisms through which physiologic or pathologic mechanical signals are transduced into a cellular response are not well understood. Defining the role of mechanosensory pathways in cartilage during OA pathogenesis may yield novel strategies or targets for the treatment of OA. The transient receptor potential vanilloid 4 (TRPV4) ion channel transduces mechanical loading of articular cartilage via the generation of intracellular calcium ion transients. Using tissue-specific, inducible Trpv4 gene-targeted mice, we demonstrate that loss of TRPV4-mediated cartilage mechanotransduction in adulthood reduces the severity of aging-associated OA. However, loss of chondrocyte TRPV4 did not prevent OA development following destabilization of the medial meniscus (DMM). These results highlight potentially distinct roles of TRPV4-mediated cartilage mechanotransduction in age-related and post-traumatic OA, and point to a novel disease-modifying strategy to therapeutically target the TRPV4-mediated mechanotransduction pathway for the treatment of aging-associated OA.

Direct reversal of glucocorticoid resistance by AKT inhibition in acute lymphoblastic leukemia.

Glucocorticoid resistance is a major driver of therapeutic failure in T cell acute lymphoblastic leukemia (T-ALL). Here, we identify the AKT1 kinase as a major negative regulator of the NR3C1 glucocorticoid receptor protein activity driving glucocorticoid resistance in T-ALL. Mechanistically, AKT1 impairs glucocorticoid-induced gene expression by direct phosphorylation of NR3C1 at position S134 and blocking glucocorticoid-induced NR3C1 translocation to the nucleus. Moreover, we demonstrate that loss of PTEN and consequent AKT1 activation can effectively block glucocorticoid-induced apoptosis and induce resistance to glucocorticoid therapy. Conversely, pharmacologic inhibition of AKT with MK2206 effectively restores glucocorticoid-induced NR3C1 translocation to the nucleus, increases the response of T-ALL cells to glucocorticoid therapy, and effectively reverses glucocorticoid resistance in vitro and in vivo.

Genetic loss of SH2B3 in acute lymphoblastic leukemia.

The SH2B adaptor protein 3 (SH2B3) gene encodes a negative regulator of cytokine signaling with a critical role in the homeostasis of hematopoietic stem cells and lymphoid progenitors. Here, we report the identification of germline homozygous SH2B3 mutations in 2 siblings affected with developmental delay and autoimmunity, one in whom B-precursor acute lymphoblastic leukemia (ALL) developed. Mechanistically, loss of SH2B3 increases Janus kinase-signal transducer and activator of transcription signaling, promotes lymphoid cell proliferation, and accelerates leukemia development in a mouse model of NOTCH1-induced ALL. Moreover, extended mutation analysis showed homozygous somatic mutations in SH2B3 in 2 of 167 ALLs analyzed. Overall, these results demonstrate a Knudson tumor suppressor role for SH2B3 in the pathogenesis of ALL and highlight a possible link between genetic predisposition factors in the pathogenesis of autoimmunity and leukemogenesis.

Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we report the presence of loss-of-function mutations and deletions of the EZH2 and SUZ12 genes, which encode crucial components of the Polycomb repressive complex 2 (PRC2), in 25% of T-ALLs. To further study the role of PRC2 in T-ALL, we used NOTCH1-dependent mouse models of the disease, as well as human T-ALL samples, and combined locus-specific and global analysis of NOTCH1-driven epigenetic changes. These studies demonstrated that activation of NOTCH1 specifically induces loss of the repressive mark Lys27 trimethylation of histone 3 (H3K27me3) by antagonizing the activity of PRC2. These studies suggest a tumor suppressor role for PRC2 in human leukemia and suggest a hitherto unrecognized dynamic interplay between oncogenic NOTCH1 and PRC2 function for the regulation of gene expression and cell transformation.

Induction of triggering receptor expressed on myeloid cells (TREM-1) in airway epithelial cells by 1,25(OH)2 vitamin D3.

The airway epithelium plays a role in host defense through the binding of innate immune receptors, which leads to the activation of inflammatory mediators, including antimicrobial peptides. The active form of vitamin D, 1,25(OH)(2)D(3), induces the expression of the antimicrobial peptide LL-37 in both myeloid cells and airway epithelial cells (AEC). Here, we demonstrate that mRNA encoding triggering receptor expressed on myeloid cells (TREM)-1 was induced up to 12-fold by 1,25(OH)(2)D(3) in normal human bronchial epithelial (NHBE) cells and in well-differentiated cultures of six airway epithelial cell lines from patients with cystic fibrosis and healthy individuals. TREM-2 and DAP12 were also expressed in airway cultures, but not induced by vitamin D. Induction occurs through a vitamin D response element identified in its proximal promoter region, and was regulated by PU.1 expressed in the AEC. Activation of TREM-1 by a cross-linking antibody led to an induction of both human ß-defensin-2 and TNF-α mRNA, demonstrating its functionality in these cells. Our results expand on the role played by the airway epithelium in innate immunity and suggest that vitamin D can modulate the innate immune defense of the airway epithelium, and could potentially be developed as an adjunctive therapy for airway infections.

ETV6 mutations in early immature human T cell leukemias.

Early immature T cell acute lymphoblastic leukemias (T-ALLs) account for ~5-10% of pediatric T-ALLs and are associated with poor prognosis. However, the genetic defects that drive the biology of these tumors remain largely unknown. In this study, analysis of microarray gene expression signatures in adult T-ALL demonstrated a high prevalence of early immature leukemias and revealed a close relationship between these tumors and myeloid leukemias. Many adult immature T-ALLs harbored mutations in myeloid-specific oncogenes and tumor suppressors including IDH1, IDH2, DNMT3A, FLT3, and NRAS. Moreover, we identified ETV6 mutations as a novel genetic lesion uniquely present in immature adult T-ALL. Our results demonstrate that early immature adult T-ALL represents a heterogeneous category of leukemias characterized by the presence of overlapping myeloid and T-ALL characteristics, and highlight the potential role of ETV6 mutations in these tumors.

Measuring antimicrobial peptide activity on epithelial surfaces in cell culture.

To more accurately assess the activity and role of epithelial cell-derived antimicrobial peptides in their native settings, it is essential to perform assays at the surfaces under relevant conditions. In order to carry this out, we utilize three-dimensional cultures of airway and gingival epithelium, which are grown at an air-liquid interface. Under these conditions, the cultures can be subjected to challenge with a variety of factors known to cause an increase in antimicrobial peptide gene expression. The functional relevance of this induction can then be assessed by quantifying antibacterial activity either directly on the surface of the cells or using the fluid secreted onto the apical surface of the cultures. The relative contribution of the peptides can also be measured by pre-incubation of the secreted fluid with specific inhibitory antibodies. Thus, a relatively inexpensive in vitro model can be used to evaluate the role of antimicrobial peptides in mucosal epithelium.