IntroductionMesenchymal stem cells (MSCs) are immunosuppressive, but we lack an understanding of how these adult stem cells are in turn affected by immune cells and the surrounding tissue environment. As MSCs have stromal functions and exhibit great plasticity, the influence of an inflamed microenvironment on their responses is important to determine. MSCs downregulate microglial inflammatory responses, and here we describe the mutual effects of coculturing mouse bone marrow MSCs with BV2 microglia in a lipopolysaccharide (LPS) inflammatory paradigm.MethodsMouse MSCs were cultured from femoral and tibial bone marrow aspirates and characterized. MSCs were cocultured with BV2 microglia at four seeding-density ratios (1:0.2, 1:0.1, 1:0.02, and 1:0.01 (BV2/MSC)), and stimulated with 1 μg/ml LPS. In certain assays, MSCs were separated from BV2 cells with a cell-culture insert to determine the influence of soluble factors on downstream responses. Inflammatory mediators including nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and chemokine (C-C motif) ligand 2 (CCL2) were measured in cocultures, and MSC and BV2 chemotactic ability determined by migration assays.ResultsWe demonstrated MSCs to increase expression of NO and IL-6 and decrease TNF-α in LPS-treated cocultures. These effects are differentially mediated by soluble factors and cell-to-cell contact. In response to an LPS stimulus, MSCs display distinct behaviors, including expressing IL-6 and very high levels of the chemokine CCL2. Microglia increase their migration almost fourfold in the presence of LPS, and interestingly, MSCs provide an equal impetus for microglia locomotion. MSCs do not migrate toward LPS but migrate toward microglia, with their chemotaxis increasing when microglia are activated. Similarly, MSCs do not produce NO when exposed to LPS, but secrete large amounts when exposed to soluble factors from activated microglia. This demonstrates that certain phenotypic changes of MSCs are governed by inflammatory microglia, and not by the inflammatory stimulus. Nonetheless, LPS appears to "prime" the NO-secretory effects of MSCs, as prior treatment with LPS triggers a bigger NO response from MSCs after exposure to microglial soluble factors.ConclusionsThese effects demonstrate the multifaceted and reciprocal interactions of MSCs and microglia within an inflammatory milieu.

Clostridium difficile is the most important cause of nosocomial infectious diarrhea in the western world. C. difficile infections are a major healthcare burden with approximately 500,000 new cases every year and an estimated annual cost of nearly $1 billion in the U.S. Furthermore, the infections are no longer restricted to health care facilities, and recent studies indicate spread of C. difficile infection to the community as well. The clinical spectrum of C. difficile infection ranges from asymptomatic colonization to severe diarrhea, fulminant colitis and death. This spectrum results from a complex interplay between bacterial virulence factors, the colonic microbiome and the host inflammatory response. The overall vigor of host inflammatory response is believed to be an important determinant of C. difficile disease severity, and a more robust immune response is associated with worse outcomes. Neutrophils are the primary cells that respond to C. difficile invasion and neutrophilic inflammation is the hallmark of C. difficile-associated disease. In this review, we will focus on the role of neutrophils (infiltration to infected tissue, pathogen clearance and resolution of inflammation) in the immuno-pathogenesis of C. difficile-associated disease (CDAD).

Severe Clostridiodes difficile infection (CDI) is life-threatening and responds poorly to treatment. Obesity is associated with development of severe CDI. Therefore, to define the mechanisms that exacerbate disease severity, we examined CDI pathogenesis in high-fat diet (HFD)-fed obese mice. Compared to control mice, HFD-fed mice failed to clear C. difficile bacteria which resulted in protracted diarrhea, weight loss and colonic damage. After infection, HFD-induced obese mice had an intestinal bile acid (BA) pool that was dominated by primary BAs which are known promoters of C. difficile spore germination, and lacked secondary BAs that inhibit C. difficile growth. Concurrently, synthesis of primary BAs from liver was significantly increased in C. difficile-infected HFD-fed mice. A key pathway that regulates hepatic BA synthesis is via feedback inhibition from intestinal Farnesoid X receptors (FXRs). Our data reveal that the proportion of FXR agonist BAs to FXR antagonist BAs in the intestinal lumen was significantly reduced in HFD-fed mice after CDI. Treatment of HFD-fed mice with an FXR agonist Obeticholic acid, resulted in decreased primary BA synthesis, fewer C. difficile bacteria and better CDI outcomes. Thus, OCA treatment holds promise as a therapy for severe CDI.

BackgroundProgression of neurodegenerative diseases occurs when microglia, upon persistent activation, perpetuate a cycle of damage in the central nervous system. Use of mesenchymal stem cells (MSC) has been suggested as an approach to manage microglia activation based on their immunomodulatory functions. In the present study, we describe the mechanism through which bone marrow-derived MSC modulate the proliferative responses of lipopolysaccharide-stimulated BV2 microglia.MethodsBV2 microglia were cultured with MSC and stimulated with 1 μg/ml lipopolysaccharide. Using an inducible nitric oxide synthase inhibitor, tritiated thymidine (3H-TdR) incorporation assay was performed to determine the role of nitric oxide in the anti-proliferative effect of MSC. We also studied apoptosis and the cell cycle of both cell types using flow cytometry and explored their cytokine profile using protein and cytometric arrays. Moreover, the role of IL-6 and TNF-α in immunomodulation was deduced using specific blocking antibodies and recombinant proteins.ResultsMSC reduces microglia proliferation upon lipopolysaccharide stimulation by 21 to 28% and modulates the levels of nitric oxide, IL-6 and TNF-α. The role of nitric oxide in conferring the anti-proliferative effect of MSC was ruled out. Furthermore, we found that MSC exert their anti-proliferative effect by restoring the percentage of BV2 cells at S and G2/M phase to levels similar to unstimulated cells. MSC undergo a G0/G1 arrest while exerting this effect. We have also identified that MSC-mediated modulation of microglia is independent of IL-6, whilst reduction of TNF-α in co-culture is critical for inhibition of microglia proliferation.ConclusionsOur study demonstrates that MSC inhibit microglia proliferation independent of nitric oxide and IL-6, although reduction of TNF-α is critical for this effect. The inhibition of proliferation is through cell cycle modulation. These findings shed light on the mechanisms of microglial immunomodulation by MSC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0149-8) contains supplementary material, which is available to authorized users.