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Neutrophils are the first responders to sites of inflammation when the intestinal epithelial barrier is breached and the gut microbiota invade. Despite current efforts in understanding the role of neutrophils in intestinal homeostasis, the complex interactions between neutrophils and intestinal epithelial cells (IECs) is still not well characterized. In this study, we demonstrated that neutrophils enhanced production of amphiregulin (AREG), a member of the EGFR ligand family, by IECs, which promoted IEC barrier function and tissue repair. Depletion of neutrophils resulted in more severe colitis in mice because of decreased AREG production by IECs upon dextran sodium sulfate (DSS) insult. Administration of AREG restored epithelial barrier function and ameliorated colitis. Furthermore, neutrophil-derived TGF-β promoted AREG production by IECs. Mechanistically, TGF-β activated MEK1/2 signaling, and inhibition of MEK1/2 abrogated TGF-β–induced AREG production by IECs. Collectively, these findings reveal that neutrophils play an important role in the maintenance of IEC barrier function and homeostasis.

Commensal flora plays important roles in the regulation of the gene expression involved in many intestinal functions and the maintenance of immune homeostasis, as well as in the pathogenesis of inflammatory bowel diseases. The microRNAs (miRNAs), a class of small, noncoding RNAs, act as key regulators in many biological processes. The miRNAs are highly conserved among species and appear to play important roles in both innate and adaptive immunity, as they can control the differentiation of various immune cells, as well as their functions. However, it is still largely unknown how microbiota regulates miRNA expression, thereby contributing to intestinal homeostasis and pathogenesis of inflammatory bowel disease. In our current study, we found that microbiota negatively regulated intestinal miR-10a expression, because the intestines, as well as intestinal epithelial cells and dendritic cells of specific pathogen-free mice, expressed much lower levels of miR-10a compared with those in germ-free mice. Commensal bacteria downregulated dendritic cell miR-10a expression via TLR–TLR ligand interactions through a MyD88-dependent pathway. We identified IL-12/IL-23p40, a key molecule for innate immune responses to commensal bacteria, as a target of miR-10a. The ectopic expression of the miR-10a precursor inhibited, whereas the miR-10a inhibitor promoted, the expression of IL-12/IL-23p40 in dendritic cells. Mice with colitis expressing higher levels of IL-12/IL-23p40 exhibited lower levels of intestinal miR-10a compared with control mice. Collectively, our data demonstrated that microbiota negatively regulates host miR-10a expression, which may contribute to the maintenance of intestinal homeostasis by targeting IL-12/IL-23p40 expression.

Activation-induced cytidine deaminase (AID) is an enzyme essential for the generation of Ab diversity in B cells and is considered to be a general gene mutator. In addition, AID expression was also implicated in the pathogenesis of human B cell malignancies and associated with poor prognosis. In this study, we report that small interfering RNA silencing of AID in plasmacytoma dramatically increased its susceptibility to immunotherapy by CTLs. AID silencing did not decrease the mutation frequencies of tumor Ag gene P1A. Gene-array analysis showed dramatically altered expression of a number of genes in AID-silenced plasmacytoma cells, and upregulation of CD200 was shown to be in favor of tumor eradication by CTLs. Taken together, we demonstrate a novel function of AID in tumor evasion of CTL therapy and that targeting AID should be beneficial in the immunotherapy of AID-positive tumors.

We report here that miR-155 and miR-125b play a role in innate immune response. LPS stimulation of mouse Raw 264.7 macrophages resulted in the up-regulation of miR-155 and down-regulation of miR-125b levels. The same changes also occurred when C57BL/6 mice were i.p. injected with LPS. Furthermore, the levels of miR-155 and miR-125b in Raw 264.7 cells displayed oscillatory changes in response to TNF-α. These changes were impaired by pretreating the cells with the proteasome inhibitor MG-132, suggesting that these two microRNAs (miRNAs) may be at least transiently under the direct control of NF-κB transcriptional activity. We show that miR-155 most probably directly targets transcript coding for several proteins involved in LPS signaling such as the Fas-associated death domain protein (FADD), IκB kinase ε (IKKε), and the receptor (TNFR superfamily)-interacting serine-threonine kinase 1 (Ripk1) while enhancing TNF-α translation. In contrast, miR-125b targets the 3′-untranslated region of TNF-α transcripts; therefore, its down-regulation in response to LPS may be required for proper TNF-α production. Finally, Eμ-miR-155 transgenic mice produced higher levels of TNF-α when exposed to LPS and were hypersensitive to LPS/d-galactosamine-induced septic shock. Altogether, our data suggest that the LPS/TNF-α-dependent regulation of miR-155 and miR-125b may be implicated in the response to endotoxin shock, thus offering new targets for drug design.

Experimental autoimmune encephalomyelitis (EAE), studied as a model for multiple sclerosis (MS), is mediated by autoreactive CD4+ T cells. MicroRNAs (miRNAs) are a class of small, noncoding RNAs that regulate gene expression posttranscriptionally. Aberrant expression of miRNAs has been implicated in various disease processes. Recent evidence indicates that miRNAs are central to immune function and play a role in T cell responsiveness to antigen stimulation. We hypothesized that certain miRNAs would be differentially expressed in mice with EAE, and that these miRNAs represent critical mediators of autoimmune disease pathology. We utilized microarray technology, followed by PCR verification, to evaluate miRNA expression level at varying times during the EAE disease course. RNA was obtained from the lymph nodes and spleen during the acute and effector phases of EAE, and compared to animals receiving adjuvant alone. We found that several miRNAs are up- or downregulated during EAE and expression is temporally distinct. These data indicate that miRNAs represent a critical component of EAE pathology, and may be useful therapeutic targets or biomarkers in MS and other autoimmune diseases. Supported by NIH grants T32 GM068412, NS 48316 and AI 064320.

Activation induced cytidine deaminase (AID) is an essential enzyme that controls the generation of antibody diversity in B cells and is considered a general gene mutator. Using transgenic T cells specific to P1A (P1CTL) to treat mice with large established plasmacytoma J558 tumors, we frequently observed mutations in P1A antigenic epitope, which lead to cancer cell evasion of destruction by P1CTL. To determine whether AID plays a role in CTL-mediated rejection of plasmacytoma, we generated AID-silenced plasmacytoma J558 cells and controls and tested whether large established AID-silenced J558 tumors could be rejected by P1CTL. Complete rejection of large established tumors by P1CTL was rare (0–10%) in mice with AID+ J558 tumors while the rejection rates were 30–90% in mice with AID-silenced J558 tumors. AID knock down did not reduce mutation rate of P1A gene, but rather caused more genetic instability and altered gene expression profile. Notably, we observed strong up-regulation of OX-2 (CD200) gene and down-regulation of CaMKII-δ gene in AID-silenced J558 cells, whose roles may be related to enhanced immune rejection of AID-silenced J558 tumors. Our results suggest that simultaneously targeting AID in CTL-targeted therapy of myeloma or other AID+ tumors may be of therapeutic value.