Active episodes of the inflammatory bowel diseases (IBD) are associated with the infiltration of large numbers of myeloid cells including neutrophils, monocytes and macrophages. The objective of this study was to systematically characterize and define the different populations of myeloid cells generated in a mouse model of chronic gut inflammation. Using the T cell transfer model of chronic colitis, we found that induction of disease was associated with enhanced production of myelopoietic cytokines (IL-17, G-CSF), increased production of neutrophils and monocytes and infiltration of large numbers of myeloid cells into the MLNs and colon. Detailed characterization of these myeloid cells revealed three major populations including Mac-1+Ly6ChighGr-1low/neg cells (monocytes), Mac-1+Ly6CintGr-1+ cells (neutrophils) and Mac-1+Ly6Clow/negGr-1low/neg leukocytes (macrophages, dendritic cells, and eosinophils). In addition, we observed enhanced surface expression of MHC class II and CD86 on neutrophils isolated from the inflamed colon when compared to neutrophils obtained from the blood, MLNs and spleen of colitic mice. Furthermore, we found that colonic neutrophils had acquired antigen-presenting cell (APC) function that enabled these granulocytes to induce proliferation of ovalbumin-specific CD4+ T cells in an antigen- and MHC class II-dependent manner. Finally, we observed a synergistic increase in pro-inflammatory cytokine and chemokine production following co-culture of T cells with neutrophils in vitro. Taken together, our data suggest that extravasated neutrophils acquire APC function within the inflamed bowel where they may perpetuate chronic gut inflammation by inducing T cell activation and proliferation as well as by enhancing production of pro-inflammatory mediators.

Maladaptive repair after AKI may lead to progressive fibrosis and decline in kidney function. Sphingosine 1-phosphate has an important role in kidney injury and pleiotropic effects in fibrosis. We investigated the involvement of sphingosine kinase 1 and 2 (SphK1 and SphK2), which phosphorylate sphingosine to produce sphingosine 1-phosphate, in kidney fibrosis induced by folic acid (FA) or unilateral ischemia-reperfusion injury. Analysis of Masson trichrome staining and fibrotic marker protein and mRNA expression 14 days after AKI revealed that wild-type (WT) and mice exhibited more kidney fibrosis than mice. Furthermore, kidneys of FA-treated WT and mice had greater immune cell infiltration and expression of fibrotic and inflammatory markers than kidneys of FA-treated mice. In contrast, kidneys of mice exhibited greater expression of and IFN--responsive genes ( and ) than kidneys of WT or mice did at this time point. Splenic T cells from untreated mice were hyperproliferative and produced more IFN- than did those of WT or mice. IFN- blocking antibody administered to mice or deletion of ( mice) blocked the protective effect of SphK2 deficiency in fibrosis. Moreover, adoptive transfer of (but not ) CD4 T cells into WT mice blocked FA-induced fibrosis. Finally, a selective SphK2 inhibitor blocked FA-induced kidney fibrosis in WT mice. These studies demonstrate that SphK2 inhibition may serve as a novel therapeutic approach for attenuating kidney fibrosis.

Clinical trials and animal studies have revealed a role for the renin-angiotensin system in the enhanced thrombus development that is associated with hypertension. Since T-lymphocytes have been implicated in the vascular dysfunction and blood pressure elevation associated with increased angiotensin II (AngII) levels, we evaluated the role of the adaptive immune system in mediating the enhanced thrombosis during AngII-induced hypertension. Light/dye-induced thrombosis was induced in cremaster arterioles of wild type (WT), immunodeficient Rag-1−/−, CD8+ or CD4+-lymphocyte-deficient, and NADPH oxidase (gp91phox) deficient mice implanted with an AngII-loaded pump for 2 weeks. Chronic AngII infusion enhanced arteriolar thrombosis in WT mice but not in Rag-1−/−, CD4+T-cell deficient, or gp91phox−/− mice. CD8+ T-cell−/−-mice exhibited partial protection. Adoptive transfer of T-cells derived from WT- or gp91phox−/−-mice into Rag-1−/− restored the prothrombotic phenotype induced by AngII. T-lymphocytes (CD4+ and, to a lesser extent, CD8+) play a major role in mediating the accelerated microvascular thrombosis associated with AngII-induced hypertension. NADPH oxidase-derived reactive oxygen species, produced by cells other T-lymphocytes, also appear critical for the AngII-enhanced, T-cell dependent thrombosis response.

The plasticity of dendritic cells (DCs) permits phenotypic modulation ex vivo by gene expression or pharmacologic agents, and these modified DCs can exert therapeutic immunosuppressive effects in vivo through direct interactions with T cells, either inducing T regulatory cells (T REG s) or causing anergy. Sphingosine 1-phosphate (S1P) is a sphingolipid and the natural ligand for five G protein-coupled receptors (S1P1, S1P2, S1P3, S1P4, and S1P5), and S1PR agonists reduce kidney ischemia-reperfusion injury (IRI) in mice. S1pr3 2/2 mice are protected from kidney IRI, because DCs do not mature. We tested the therapeutic advantage of S1pr3 2/2 bone marrow-derived dendritic cell (BMDC) transfers in kidney IRI. IRI produced a rise in plasma creatinine (PCr) levels in mice receiving no cells (NCs) and mice pretreated with wild-type (WT) BMDCs. However, S1pr3 2/2 BMDC-pretreated mice were protected from kidney IRI. S1pr3 2/2BMDC-pretreated mice had significantly higher numbers of splenic T REG s compared with NC and WT BMDC-pretreated mice. S1pr3 2/2 BMDCs did not attenuate IRI in splenectomized, Rag-1 2/2 , or CD11c + DC-depleted mice. Additionally, S1pr3 2/2 BMDC-dependent protection required CD169 + marginal zone macrophages and the macrophage-derived chemokine CCL22 to increase splenic CD4 + Foxp3 + T REG s.Pretreatment with S1pr3 2/2 BMDCs also induced T REG -dependent protection against IRI in an allogeneic mouse model. In summary, adoptively transferred S1pr3 2/2 BMDCs prevent kidney IRI through interactions within the spleen and expansion of splenic CD4 + Foxp3 + T REG s. We conclude that genetically induced deficiency of S1pr3 in allogenic BMDCs could serve as a therapeutic approach to prevent IRI-induced AKI.

We have previously shown that polyclonal natural IgM protects mice from renal IRI by inhibiting the reperfusion inflammatory response. We hypothesized that a potential mechanism involved IgM modulation of dendritic cells as we observed high IgM binding to splenic DC. To test this hypothesis, we pre-treated BMDC with polyclonal murine or human IgM prior to LPS activation and demonstrate that 0.5 × 106 IgM/LPS pretreated BMDC, when injected into WT-B6 mice, 24 hours before renal ischemia, protect mice from developing renal IRI. We show that this switching of LPS activated BMDC to a regulatory phenotype requires modulation of BMDC function that is mediated by IgM binding to non-apoptotic BMDC receptors. Regulatory BMDC require IL-10 and PD1 as well as downregulation of CD40 and p65NF-κB phosphorylation to protect in renal IRI. Blocking the PD1 ligand binding site just before intravenous injection of IgM/LPS pretreated BMDC or using IL-10ko BMDC fails to induce protection. Similarly, IgM/LPS pretreated BMDC are rendered non-protective by increasing CD40 expression and phosphorylation of p65NF-κB. How IgM/LPS regulatory BMDC suppress in-vivo ischemia induced innate inflammation remains to be determined. However, we show that suppression is dependent on other in-vivo regulatory mechanisms in the host i.e. CD25+ T cells, B cells, IL10 and circulating IgM. There was no increase in Foxp3+ Tregs in the spleen either before or after renal IRI. Collectively, these findings show that natural IgM anti-leucocyte antibodies can switch BMDC to a regulatory phenotype despite the presence of LPS that ordinarily induces BMDC maturation.