The expression level was calculated using the 2?Ct cycle threshold method after normalization to the endogenous U6 RNA as an internal control. Ca2+ Assay Intracellular calcium levels in phagocytes were measured using the cell-based Fluo-4 NW Calcium Assay kit according Risarestat to the manufacturers protocol (Molecular Probes, Eugene, OR, CCNB1 USA). ELISA Specific ELISA kits were used to measure the levels of S100A8, S100A9 (MyBioSource, San Diego, CA, USA) and S100A8/A9 heterodimer (R&D Systems, Minneapolis, MN, USA). protein translocates from the cytosol to nucleus in Gr1+CD11b+ MDSCs during late sepsis and promotes expression of miR-21 and miR-181b immune repressor mediators. We further provide support of this immunosuppression pathway in human sepsis. This study may inform a new therapeutic target for improving sepsis outcome. fertilization of C57BL/6NJ oocytes to reconstitute the strain (work performed by TransViragen, Chapel Hill, NC, USA). Heterozygous animals were intercrossed to generate homozygous (?/?) mutant animals for study. The mice were bred and housed in a pathogen-free facility in the Division of Laboratory Animal Resources. Wild-type male C57BL/6J mice, 8C10?weeks were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and used as controls, and were acclimated to the new environment for a week before surgery. All experiments were conducted in accordance with National Institutes of Health guidelines and were approved by the East Tennessee State University Animal Care and Use Committee. Polymicrobial Sepsis Polymicrobial sepsis was induced in male wild-type and S100A9 knockout mice, 8C10 week old, by cecal ligation and puncture (CLP) as described previously (26). Briefly, mice were anesthetized inhalation with 2.5% isoflurane (Abbott Risarestat Laboratories, Abbott Park, IL, USA). A midline abdominal incision was made and the cecum was exteriorized, ligated distal to the ileocecal valve, and then punctured twice with a 23-gauge needle. A small amount of feces was extruded into the abdominal cavity. The abdominal wall and skin were sutured in layers with 3-0 silk. Sham-operated mice were treated identically except that the cecum was neither ligated nor punctured. Mice received (i.p.) 1?ml lactated Ringers plus 5% dextrose for fluid resuscitation. To induce sepsis that develops into early and late phases, mice were subcutaneously administered antibiotic (Imipenem; 25?mg/kg body weight) or an equivalent volume of 0.9% saline. To establish intra-abdominal infection and approximate the clinical Risarestat condition of early human sepsis where there is a delay between the onset of sepsis and the delivery of therapy (27), injections of Imipenem were given at 8 and 16?h after CLP. Based on our experience, these levels of injury and manipulation create prolonged infections with high mortality (~60C70%) during the late/chronic phase (26). The presence of early sepsis was confirmed by transient systemic bacteremia and elevated cytokine levels in the first 5?days after CLP. Late/chronic sepsis (after day 5) was confirmed by enhanced peritoneal bacterial overgrowth and reduced circulating pro-inflammatory cytokines. Table S1 in Supplementary Material includes the CLP mice that were used in the study. Sepsis Patients Patients 18?years of age or Risarestat older who were admitted to Johnson City Medical Center and Franklin Woods Hospital in Johnson City, Tennessee, and who Risarestat were diagnosed with sepsis or septic shock were included in the study. Sepsis was defined as the presence of suspected or documented infection with at least two of the following criteria: core temperature 38C or 36C; heart rate 90?beats/min; respiratory rate 20?breaths/min or arterial blood partial pressure of carbon dioxide 32?mmHg; or white blood cell count 12,000 cells/mm3 or 4,000/mm3. Septic shock was defined as sepsis with persisting hypotension requiring vasopressors to maintain MAP 65?mmHg and having a serum lactate 2?mmol/L despite adequate volume resuscitation (28). Patients presented with infections related to Gram-negative or Gram-positive bacteria. The primary infection included urinary tract infection, blood stream infection, and respiratory tract infection. Patients had at least 1 comorbid condition, including nephropathy, psoriasis, splenectomy, colon cancer, or pulmonary aspergillosis. Patients with leukopenia due to chemotherapy or glucocorticoid therapy or HIV infection were excluded from the study. Patients were divided into two categories: early sepsis and late sepsis, relative to the day of sepsis diagnosis. The early septic group included patients within 1C5?days of sepsis diagnosis. Those who have been septic for.