There is an increasing interest in using microRNAs (miRNA) as biomarkers in autoimmune diseases. They are easily accessible in many body fluids but it is controversial if they are circulating freely or are encapsulated in microvesicles, particularly exosomes. We investigated if the majority of miRNas in serum and saliva are free-circulating or concentrated in exosomes. Exosomes were isolated by ultracentrifugation from fresh and frozen human serum and saliva. The amount of selected miRNAs extracted from the exosomal pellet and the exosome-depleted serum and saliva was compared by quantitative RT-PCR. Some miRNAs tested are ubiquitously expressed, others were previously reported as biomarkers. We included miRNAs previously reported to be free circulating and some thought to be exosome specific. The purity of exosome fraction was confirmed by electronmicroscopy and western blot. The concentration of miRNAs was consistently higher in the exosome pellet compared to the exosome-depleted supernatant. We obtained the same results using an equal volume or equal amount of total RNA as input of the RT-qPCR. The concentration of miRNA in whole, unfractionated serum, was between the exosomal pellet and the exosome-depleted supernatant. Selected miRNAs, which were detectable in exosomes, were undetectable in whole serum and the exosome-depleted supernantant. Exosome isolation improves the sensitivity of miRNA amplification from human biologic fluids. Exosomal miRNA should be the starting point for early biomarker studies to reduce the probability of false negative results involving low abundance miRNAs that may be missed by using unfractionated serum or saliva.

MicroRNAs (miRs) seem to mediate renal fibrosis in several renal diseases, with some miRs having profibrotic effects and others having opposing effects. Although differential expression of certain miRs has been described in lupus nephritis, it is unknown whether miRs contribute to fibrosis or could serve as biomarkers of specific histologic manifestations of lupus nephritis. Here, we compared miR expression in kidney biopsies from patients with lupus nephritis and identified miR-150 as the most differentially expressed miR in kidneys with high chronicity (chronicity index [CI] $4); miR-150 positively correlated with chronicity scores and the expression of profibrotic proteins. Overexpression of miR-150 significantly reduced expression of the antifibrotic protein suppressor of cytokine signaling 1 (SOCS1) and upregulated profibrotic proteins in both proximal tubular and mesangial cells. Directly targeting SOCS1 with a small interfering RNA produced similar results. Furthermore, TGF-b1 induced miR-150 expression, decreased SOCS1, and increased profibrotic proteins in proximal tubular cells and podocytes; a miR-150 inhibitor reversed these changes, suggesting that the profibrotic effects of TGF-b1 are, at least in part, mediated by miR-150. Consistent with these in vitro observations, biopsies with high miR-150 and high CI exhibited substantial expression of TGF-b1, reduced SOCS1, and an increase in profibrotic proteins. In summary, miR-150 is a promising quantitative renal biomarker of kidney injury in lupus nephritis. Our results suggest that miR-150 promotes renal fibrosis by increasing profibrotic molecules through downregulation of SOCS1.

The gut microbiome participates in numerous physiologic functions and communicates intimately with the host immune system. Antimicrobial peptides are critical components of intestinal innate immunity. We report a prominent role for antimicrobials secreted by pancreatic acinari in shaping the gut microbiome that is essential for intestinal innate immunity, barrier function, and survival. Deletion of the Ca2+ channel Orai1 in pancreatic acini of adult mice resulted in 60–70% mortality within three weeks. Despite robust activation of the intestinal innate immune response, mice lacking acinar Orai1 exhibited intestinal bacterial outgrowth and dysbiosis, ultimately causing systemic translocation, inflammation, and death. While digestive enzyme supplementation was ineffective, treatments constraining bacterial outgrowth (purified liquid diet, broad-spectrum antibiotics), rescued survival, feeding, and weight gain. Pancreatic levels of cathelicidin-related antimicrobial peptide (CRAMP) were reduced, and supplement of synthetic CRAMP prevented intestinal disease. These findings reveal a critical role for antimicrobial pancreatic secretion in gut innate immunity.