IL10 receptor (IL10R)-deficient mice develop spontaneous colitis and similarly, patients with loss-of-function mutations in IL10R develop severe infant-onset inflammatory bowel disease (IBD). Loss of IL10R signaling in mouse and human macrophages is associated with increased production of interleukin 1 beta (IL1B). We demonstrated that innate immune production of IL1B mediates colitis in IL10R-deficient mice. Transfer of Il1r1−/− CD4+ T cells into Rag1−/−/Il10rb−/− mice reduced the severity of their colitis (compared to mice that received CD4+ T cells that express IL1R), accompanied by decreased production of interferon gamma, tumor necrosis factor, and IL17A. In macrophages from mice without disruption of IL10R signaling or from healthy humans (controls), incubation with IL10 reduced canonical activation of the inflammasome and production of IL1B through transcriptional and post-translational regulation of NLRP3. Lipopolysaccharide (LPS) and adenosine triphosphate stimulation of macrophages from Il10rb−/− mice or IL10R-deficient patients increased production of IL1B. Moreover, in human IL10R-deficient macrophages, LPS stimulation alone increased IL1B secretion via non-canonical, caspase 8-dependent activation of the inflammasome. We treated 2 IL10-receptor deficient patients with severe and treatment-refractory infant-onset IBD with the IL1 receptor antagonist anakinra. Both patients had marked clinical, endoscopic, and histologic responses after 4–7 weeks. This treatment served as successful bridge to allogeneic hematopoietic stem cell transplantation in 1 patient. Our findings indicate that loss of IL10 signaling leads to intestinal inflammation, at least in part, through increased production of IL1 by innate immune cells, leading to activation of CD4+ T cells. Agents that block IL1 signaling might be used to treat patients with IBD resulting from IL10R deficiency.

Inflammatory bowel disease (IBD) continues to increase in prevalence in industrialized countries. Major complications of IBD include formation of fibrotic strictures, fistulas, reduced absorptive function, cancer risk, and the need for surgery. In other chronic gastrointestinal disease models, stiffness has been shown to precede fibrosis; therefore, stiffness may be a reasonable indicator of progression toward stricture formation in IBD patients. Herein, we seek to quantify tissue stiffness and characterize fibrosis in patients with IBD and to compare mechanical properties of unaffected human tissue to common animal species used for IBD studies. Inflamed and unaffected tissue from IBD patients and unaffected tissue from mice, pigs, and cows were indented using a custom device to determine the effective stiffness. Histology was performed on matched tissues, and total RNA was isolated from IBD tissue samples and used for gene expression analysis of pro-fibrotic genes. We observed an increase in the effective stiffness (steady-state modulus, SSM) (p < 0.0001) and increased expression of the collagen type I gene (COL1A1, p = 0.01) in inflamed tissue compared to unaffected areas in our IBD patient cohort. We also found that increased staining of collagen fibers in submucosa positively correlated with SSM (p = 0.093). We determined that unaffected animal bowel stiffness is significantly greater than similar human tissues, suggesting additional limitations on animal models for translational investigations regarding stiffness-related hypotheses. Taken together, our data support development of tools for evaluation of bowel stiffness in IBD patients for prognostic applications that may enable more accurate prediction of those who will develop fibrosis and more precise prescription of aggressive therapies.

Human monocytes express known markers of dopamine synthesis, storage and clearance, including dopamine transporter (DAT), tyrosine hydroxylase (TH), all subtypes of dopamine receptors and vesicular monoamine transporter 2 (VMAT2). Immunohistochemical and immunofluorescent methodologies have traditionally been employed to determine DAT and TH expression in the CNS, their detection in the blood and specifically in the peripheral monocytes has not been studied by flow cytometry. Flow cytometry assays are widely used in medicine and in basic, preclinical or clinical research to quantify physical and chemical characteristics of target cell populations. Here, we have established a highly sensitive and reproducible flow cytometry panel to detect and quantify DAT and TH expression in freshly isolated or cryopreserved human peripheral monocytes. In healthy humans (n=41 biological replicates), we show baseline DAT and TH expressing monocytes constitute ~12% of the peripheral blood mononuclear cell (PBMC) fraction when examined in fresh isolation from whole blood. Using an identical flow cytometry panel, we found that cryopreservation of PBMCs using multiple techniques resulted in altered PBMC populations as compared to fresh isolation and relative to one another. Among these, we identified an optimum cryopreservation method for detecting TH and DAT in cryopreserved PBMCs. Our data provide a sensitive and reproducible approach to examine dopamine signaling in peripheral human immune cells. This approach can be applied to study peripheral dopamine signaling under healthy and potentially under disease conditions. The use of dopamine signaling could also be explored as a technique to monitor therapeutic interventions particularly those targeting DAT and TH in the periphery.

Glioblastoma (GBM) exhibits populations of cells that drive tumorigenesis, treatment resistance, and disease progression. Cells with such properties have been described to express specific surface and intracellular markers or exhibit specific functional states, including being slow-cycling or quiescent with the ability to generate proliferative progenies. In GBM, each of these cellular fractions was shown to harbor cardinal features of cancer stem cells (CSCs). In this study, we focus on the comparison of these cells and present evidence of great phenotypic and functional heterogeneity in brain cancer cell populations with stemness properties, especially between slow-cycling cells (SCCs) and cells phenotypically defined based on the expression of markers commonly used to enrich for CSCs. Here, we present an integrative analysis of the heterogeneity present in GBM cancer stem cell populations using a combination of approaches including flow cytometry, bulk RNA sequencing, and single cell transcriptomics completed with functional assays. We demonstrated that SCCs exhibit a diverse range of expression levels of canonical CSC markers. Importantly, the property of being slow-cycling and the expression of these markers were not mutually inclusive. We interrogated a single-cell RNA sequencing dataset and defined a group of cells as SCCs based on the highest score of a specific metabolic signature. Multiple CSC groups were determined based on the highest expression level of CD133, SOX2, PTPRZ1, ITGB8, or CD44. Each group, composed of 22 cells, showed limited cellular overlap, with SCCs representing a unique population with none of the 22 cells being included in the other groups. We also found transcriptomic distinctions between populations, which correlated with clinicopathological features of GBM. Patients with strong SCC signature score were associated with shorter survival and clustered within the mesenchymal molecular subtype. Cellular diversity amongst these populations was also demonstrated functionally, as illustrated by the heterogenous response to the chemotherapeutic agent temozolomide. In conclusion, our study supports the cancer stem cell mosaicism model, with slow-cycling cells representing critical elements harboring key features of disseminating cells.

Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff). Proteins were submitted to a Sephacryl S200 column, and fractions were pooled based on the fraction pattern. Each pool was analyzed for protein concentration and tested for its suppressive effect on PMN/sperm binding. Protein pools with biological activity were submitted to ion-exchange chromatography (diethylaminoethyl [DEAE] Sephadex column) with equilibration buffers containing 0.1-0.5M NaCl. Eluants were pooled, analyzed for protein concentration, and tested for suppressive effects on PMN/sperm binding. Protein distribution and purity were determined by one- and two-dimensional SDS-PAGE, and the purified protein was submitted for sequence analysis and identification. This protein was identified as equine CRISP3 and was confirmed by Western blotting. Suppression of PMN/sperm binding by CRISP3 and seminal plasma was confirmed by flow cytometry (22.08% ± 3.05% vs. 2.06% ± 2.02% vs. 63.09% ± 8.67 for equine seminal plasma, CRISP3, and media, respectively; P < 0.0001). It was concluded that CRISP3 in seminal plasma suppresses PMNs/sperm binding, suggesting that CRISP3 regulates sperm elimination from the female reproductive tract.