Bone marrow stromal cells attenuate sepsis via prostaglandin E2–dependent reprogramming of host macrophages to increase their interleukin-10 production

Sepsis causes over 200,000 deaths yearly in the US; better treatments are urgently needed. Administering bone marrow stromal cells (BMSCs—also known as mesenchymal stem cells) to mice before or shortly after inducing sepsis by cecal ligation and puncture reduced mortality and improved organ function. The beneficial effect of BMSCs was eliminated by macrophage depletion or pretreatment with antibodies specific for interleukin-10 (IL-10) or IL-10 receptor. Monocytes and/or macrophages from septic lungs made more IL-10 when prepared from mice treated with BMSCs versus untreated mice. Lipopolysaccharide (LPS)-stimulated macrophages produced more IL-10 when cultured with BMSCs, but this effect was eliminated if the BMSCs lacked the genes encoding Toll-like receptor 4, myeloid differentiation primary response gene-88, tumor necrosis factor (TNF) receptor-1a or cyclooxygenase-2. Our results suggest that BMSCs (activated by LPS or TNF-α) reprogram macrophages by releasing prostaglandin E2 that acts on the macrophages through the prostaglandin EP2 and EP4 receptors. Because BMSCs have been successfully given to humans and can easily be cultured and might be used without human leukocyte antigen matching, we suggest that cultured, banked human BMSCs may be effective in treating sepsis in high-risk patient groups

Bone marrow stromal cells attenuate sepsis via prostaglandin E2— dependent reprogramming of host macrophages to increase their interleukin-10 production

Sepsis causes over 200,000 deaths yearly in the US; better treatments are urgently needed. Administering bone marrow stromal cells (BMSCs—also known as mesenchymal stem cells) to mice before or shortly after inducing sepsis by cecal ligation and puncture reduced mortality and improved organ function. The beneficial effect of BMSCs was eliminated by macrophage depletion or pretreatment with antibodies specific for interleukin-10 (IL-10) or IL-10 receptor. Monocytes and/ or macrophages from septic lungs made more IL-10 when prepared from mice treated with BMSCs versus untreated mice. Lipopolysaccharide (LPS)-stimulated macrophages produced more IL-10 when cultured with BMSCs, but this effect was eliminated if the BMSCs lacked the genes encoding Toll-like receptor 4, myeloid differentiation primary response gene-88, tumor necrosis factor (TNF) receptor-1a or cyclooxygenase-2. Our results suggest that BMSCs (activated by LPS or TNF-α) reprogram macrophages by releasing prostaglandin E2 that acts on the macrophages through the prostaglandin EP2 and EP4 receptors. Because BMSCs have been successfully given to humans and can easily be cultured and might be used without human leukocyte antigen matching, we suggest that cultured, banked human BMSCs may be effective in treating sepsis in high-risk patient groups.Sepsis, a serious medical condition that affects 18 million people per year worldwide, is characterized by a generalized inflammatory state caused by infection. Widespread activation of inflammation and coagulation pathways progresses to multiple organ dysfunction, collapse of the circulatory system (septic shock) and death. Because as many people die of sepsis annually as from acute myocardial infarction1, a new treatment regimen is desperately needed. In the last few years, it has been discovered that BMSCs are potent modulators of immune responses2-5. We wondered whether such cells could bring the immune response back into balance, thus attenuating the underlying pathophysiology that eventually leads to severe sepsis, septic shock and death6,7. As a model of sepsis, we chose cecal ligation and puncture (CLP), a procedure that has been used for more than two decades8. This mouse model closely resembles the human disease: it has a focal origin (cecum), is caused by multiple intestinal organisms, and results in septicemia with release of bacterial toxins into the circulation. With no treatment, the majority of the mice die 24-48 h postoperatively. Originally published Nature Medicine, Vol. 15, No. 1, Jan 200

Lactiplantibacillus plantarum dfa1 Outperforms Enterococcus faecium dfa1 on Anti-Obesity in High Fat-Induced Obesity Mice Possibly through the Differences in Gut Dysbiosis Attenuation, despite the Similar Anti-Inflammatory Properties

Fat reduction and anti-inflammation are commonly claimed properties of probiotics. Lactiplantibacillus plantarum and Enterococcus faecium were tested in high fat-induced obesity mice and in vitro experiments. After 16 weeks of probiotics, L. plantarum dfa1 outperforms E. faecium dfa1 on the anti-obesity property as indicated by body weight, regional fat accumulation, serum cholesterol, inflammatory cytokines (in blood and colon tissue), and gut barrier defect (FITC-dextran assay). With fecal microbiome analysis, L. plantarum dfa1 but not E. faecium dfa1 reduced fecal abundance of pathogenic Proteobacteria without an alteration in total Gram-negative bacteria when compared with non-probiotics obese mice. With palmitic acid induction, the condition media from both probiotics similarly attenuated supernatant IL-8, improved enterocyte integrity and down-regulated cholesterol absorption-associated genes in Caco-2 cell (an enterocyte cell line) and reduced supernatant cytokines (TNF-α and IL-6) with normalization of cell energy status (extracellular flux analysis) in bone-marrow-derived macrophages. Due to the anti-inflammatory effect of the condition media of both probiotics on palmitic acid-activated enterocytes was neutralized by amylase, the active anti-inflammatory molecules might, partly, be exopolysaccharides. As L. plantarum dfa1 out-performed E. faecium dfa1 in anti-obesity property, possibly through the reduced fecal Proteobacteria, with a similar anti-inflammatory exopolysaccharide; L. plantarum is a potentially better option for anti-obesity than E. faecium

Lack of oral epithelial tongue migration in K14CreRac1^F/F mice.

<p>(A) Immunofluorescence for cytokeratins 10 and 14 (K10/14, red), fibrinogen (green) and DNA staining (Hoechst 33342-blue) delineating cellular nuclei of control tissue sections staining the oral mucosa. Two days after the oral wound the epithelial tongue is visible migrating over the fibrin(ogen) clot (green) and the oral mucosa is healed by day 3.5. (B) Tissue sections from K14Cre<i>Rac1</i>^F/F mice stained as above show complete impairment of oral mucosa migration and maintenance of the punch biopsy trajectory evident by the fibrin(ogen) staining by day 3.5 (green). Note that high magnification (20x) of K10/14 staining show progressive loss of the well defined basal layer of the oral mucosa and absence of a epithelial tongue (yellow arrows).</p

Impairment of human oral keratinocytes migration and proliferation after knock down of Rac1.

<p>(A) The human oral keratinocyte cell line (NOK-SI) was transfected with siRNAs and knockdown of Rac1 was confirmed by Western blot analyses of Rac1 in cellular lysates 72 h after transfection of two different targeting siRNAs (siRNA#1 and siRNA#2). A non-targeting siRNA oligonucleotide (siControl) and untransfected cells (Control) were used as controls. Scratch wound assay in NOK-SI cell line after control and Rac1 siRNA#2. Scratches were generated after cell confluence. <i>In vitro</i> cell migration and wound closure were assessed every 12 h. Representative pictures of the control and Rac1 siRNA#2 transfected cell cultures at the indicated time after the initial scratch are depicted. (B) Top graphic, areas of migration were measured (dotted line from figure A) in multiple wells and represented at the indicated time. The lower graphic shows cell proliferation of NOK-SI transfected with siRNA#2 against Rac1. Incorporation of [³H]thymidine in cells stimulated with EGF (+) (30 ng/ml) or left untreated (-) was measured by the accumulation of radioactivity in cellular DNA, and represented as the average of CPM± s.e.m. in triplicate samples from a representative experiment that was repeated three times (***p<0.001; *p<0.05). (C) GST pull-down of active Rac1 was performed using NOK-SI stimulated with EGF. Cell lysates were incubated with GST-PAK-N for 30 min to affinity precipitate active Rac1. PAK-bound Rac1 and total Rac1 in the corresponding total lysates were analyzed by Western blotting with a monoclonal antibody against Rac1. Total and phospho-specific antibodies were used to detect two downstream targets of Rac1, JNK and PAK1 and their corresponding phosphorylated species. (D) NOK-SI cells were transfected with Rac1 siRNA (siRNA#2) or control siRNA (siControl) and stimulated with EGF (30 ng/ml). Western blot analysis of the Rac1 downstream targets JNK and PAK1 show absence of basal p-JNK and 3 min after EGF stimulation compared to control that show detectable basal levels of p-JNK that increase after EGF exposure. p-PAK1 also show reduced basal levels and a delayed accumulation after activation by EGF as compared to control cell lysates. GAPDH was used as loading controls. In C and D.</p

Rac1 excision leads to decreased cell proliferation and limited expression of cytokeratin 6, a stress and growth-related marker, in the skin and oral mucosa after wounding.

<p>(A) Impairment of epithelial cell proliferation in the wounded area of the skin and oral mucosa of K14Cre Rac1^F/F mice. Sections of epidermis adjacent to wounds healing skin (6 days after injury) and oral mucosa wounds (3.5 days after wounding) from of K14Cre Rac1^F/F and control mice were subjected to Ki-67 staining as a marker of cell proliferation. Both anatomical sites of the K14Cre Rac1^F/F mice show clear reduction in the proliferation capacity. Bar chart represents the total number of Ki-67 positive cells in the skin and oral cavity of K14Cre Rac1^F/F and control mice (**p<0.01, * p<0.05). (B) Normal interfollicular skin does not express detectable levels of cytokeratin 6 (K6). The epithelial tongue adjacent to the wound area from normal skin (control) show remarkable up regulation of K6 in all the epithelial layers after 6 days of injury. K14Cre Rac1^F/F mice fail to express K6 in the basal layer of the epithelial tongue from the skin (yellow arrows in the high magnification insert). (C) Normal oral mucosa does not express K6 during homeostasis (Normal). Expression of K6 in the oral mucosa epithelial tongue of control mice is evident at day 2 and 3.5 after injury. Expression pattern of K6 in the oral mucosa wound healing follows the skin pattern. K14Cre Rac1^F/F mice show lack of K6 staining in the basal layer of the oral mucosa adjacent to the wound site as depicted (yellow arrowhead).</p

Deficient epithelial tongue migration in K14CreRac1^F/F mice.

<p>(A) Control mice. Immunofluorescence for cytokeratins 10 and 14 (K10/14, red) reveals the epidermal layer of the skin and the epithelial tongue migrating underneath the fibrin clot at day 6 (Green). DNA staining (Hoechst 33342-blue) delineates cellular nuclei. Note the extensive migration of the epithelial tongue in merged figure (high Magnification-20x). (B) K14Cre<i>Rac1</i>^F/F conditional knockout mice stained for cytokeratins 10 and 14 (K10/14, red) show a more limited migration of the epithelial tongue by day 6 after injury. The deficient migration of the epithelial tongue is visualized in the merged figure (High magnification-20x).</p