The role of equine seminal plasma derived cysteine rich secretory protein 3 (CRISP3) in the interaction between polymorphonuclear neutrophils (PMNs) and populations of viable or dead spermatozoa, and bacteria.

Breeding-induced endometritis is a physiological reaction to clear the uterus from excess spermatozoa and bacteria after breeding. Cysteine rich secretory protein 3 in seminal plasma (spCRISP3) protects spermatozoa from binding and destruction by uterine PMNs, but it is not clear if this involves all sperm and bacteria, or if it is selective to a sub-population of live sperm. The objective of this report was to determine if spCRISP3 (1) is selective in its suppression of PMN-binding to sperm based on viability of spermatozoa, (2) protects bacteria from binding to PMNs, and (3) to determine the localization pattern of spCRISP3 on viable and dead sperm. Semen was collected from five stallions and each ejaculate was divided into (1) live and (2) snap frozen (dead) sperm. Two distinct sperm populations were confirmed by DNA fragmentation and membrane integrity assays. CRISP3 was purified from pooled seminal plasma, and binding of PMNs (isolated from peripheral blood) to the two sperm populations and E. coli was evaluated with flow cytometry in the presence of spCRISP3. In addition, localization of spCRISP3 on live and dead spermatozoa was determined by immunocytochemistry. Comparisons between treatments were analyzed using a one-way-ANOVA and Bonferroni's comparison test, or Kruskal-Wallis ANOVA if not normally distributed. spCRISP3 significantly suppressed binding of PMNs to live spermatozoa (p < 0.0001) but had no effect on dead sperm or bacteria (p > 0.05). Immunocytochemistry confirmed binding of spCRISP3 to live, but not dead spermatozoa. It was concluded that a selective interaction between spCRISP3 and live spermatozoa may be part of a biological mechanism that allows safe transport of viable spermatozoa to the oviducts, while enabling dead spermatozoa and bacteria to be eliminated in a timely fashion after breeding.

Quantitative assessment of intestinal stiffness and associations with fibrosis in human inflammatory bowel disease.

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.

Interleukin 1ß Mediates Intestinal Inflammation in Mice and Patients With Interleukin 10 Receptor Deficiency.

Interleukin 10 receptor (IL10R)-deficient mice develop spontaneous colitis and, similarly, patients with loss-of-function mutations in IL10R develop severe infant-onset inflammatory bowel disease. Loss of IL10R signaling in mouse and human macrophages is associated with increased production of interleukin 1ß. We demonstrated that innate immune production of IL1ß 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-α, 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 IL1ß through transcriptional and post-translational regulation of NLRP3. Lipopolysaccharide and adenosine triphosphate stimulation of macrophages from Il10rb-/- mice or IL10R-deficient patients resulted in increased production of IL1ß. Moreover, in human IL10R-deficient macrophages, lipopolysaccharide stimulation alone triggered IL1ß secretion via non-canonical, caspase 8-dependent activation of the inflammasome. We treated 2 IL10R-deficient patients with severe and treatment-refractory infant-onset inflammatory bowel disease 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 inflammatory bowel disease resulting from IL10R deficiency.

The differential frequency of Lineage(-)CRTH2(-)CD45(+)NKp44(-)CD117(-)CD127(+)ILC subset in the inflamed terminal ileum of patients with Crohn's disease.

Deregulation of various components of the immune system has been reported in the inflamed gut of Crohn's disease (CD) patients. Innate lymphoid cells (ILCs) are novel innate effector lymphocytes which can rapidly respond to danger signals, from invading pathogens or tissue damage, to maintain homeostasis, especially along the mucosal surfaces. The purpose of this study is to compare composition of the intestinal ILCs subsets of CD patients with differential inflammatory conditions of the terminal ileum, which are marked by distinct histological appearances and mucosal profiles of cytokines. We observed alterations in the frequency of Lineage(-)CRTH2(-)CD45(+)NKp44(-)CD117(-)CD127(+)ILC subset in the inflamed terminal ileum.