Preliminary Study On The Role Of Skeleton In The Pathogenesis Of Sepsis And Its Mechanism

Sepsis is the most common clinical scenario in critically ill patients, resulting in215,000(9.3%) deaths from all causes in the United States annually. Furthermore, septic shock,defined as severe sepsis accompanied with hypotension that cannot be reversed with fluidresuscitation, is the most common cause of the death in the intensive care unit (ICU). Currenttherapeutic strategies for critical sepsis consist of treatment of the underlying condition,eradication of infection, improving immunity, organ support, and maintaining metabolicequilibrium.However, clinical trials still do not demonstrate their favoable effects. Adequateclinical researches revealed that immune dysfunction, hematopoietic system disordersincluding anemia and thrombocytopenia, abnormal metabolism such as hyperglycemia andinsulin resistance would harmfully affect the process and outcome of sepsis.Skeleton is a multifunctional organ with a precise dynamic balance maintained mainlyby osteoclasts (OCs) and osteoblasts (OBs). Besides providing structural integrity, movement,locomotion, and protection of internal organs, skeleton also plays a critical role in regulatinghematopoiesis, immune activity, and metabolism.Hematopoietic stem cells (HSCs) have multiple differentiation potential, producingalmost all hematopoietic lineages as well as nonhematopoietic tissues such as kidney, liver,brain, and heart. HSCs have thus been used to treat hematological disorders and organdysfunction that frequently occur during sepsis, and have been shown to correlate closely withoutcome. It is now clear that the bone with its enclosing bone marrow exerts essentialregulatory influences on HSCs via OBs and OCs. OBs and other cells form a special sitetermed "niche," where HSCs are resided in. Thus OBs can regulate the proliferation,differentiation, and survival of HSCs. Several studies have revealed the number of HSCs is animportant limiting factor for the prognosis and treatment outcome of multiple diseasesincluding critical illness. OBs and OCs also play an essential role in HSC mobilization intothe circulation, where they perform their functions. Additionally, skeleton also plays an essential role in regulating the immune system[59].The bone marrow provides a site for early and end stage lymphocyte development.Furthermore, bone is also considered as an endocrine organ that can secrete a variety ofhormones, growth factors, and cytokines, such as leptin, follistatin, osteocalcin, insulin-likegrowth factor-Ⅰ (IGF-I), IL-1, and IL-6. Among these molecules, osteocalcin, a matureOBs-specific molecule, has a critical role in regulating glucose and energy metabolism. Allthe above-mentioned fnction of bone tissue led us to explore whether and how bone isinvolved in the pathogenesis of sepsis.To explore the role of skeleton in sepsis, we have done two experiemnts. Firstly, whetherthe levels of specific biomarkers of bone in serum including bone alkaline phosphatase(BALP), osteocalcin (OC) and beta carboxy-terminal peptide of type I collagen (CTX) can beused to assess the severity of neonatal infection. Secondly, we used mouse model withdysregulated bone metabolism to explore whether and how bone affect the pathogenesis ofsepsis.MethodsPart Ⅰ The serum level of Bone Alkaline Phosphatase can be used to assess theillness severity of infected neonates in the Neonatal Intensive Care Unit1. Sixty-three neonates in the NICU were enrolled in this study. The neonates weredivided into infected (n=33) and non-infected groups (n=30).2. The scores for neonatal acute physiology-perinatal extension (SNAPPE) werecalculated and interleukin-6(IL-6), procalcitonin (PCT), BALP, OC and CTX were measuredin the neonates with or without infections, and in the infected neonates pre-andpost-treatment.Part Ⅱ Abnormal bone resulting from osteoblast-specific deletion of FGFR1affectsthe outcome of septic mice by mediating mobilization of EPC into peripheral blood viaits regulation of SDF-1α level1. Sepsis models generated by cecal ligation puncture (CLP) were used to compare withsurvival rates between mice with (Fgfr1^-/-) or without (Fgfr1fl/fl) osteoblast-specific deletion ofFGFR1.2. After being peritoneally injected with LPS, the number of circulating endothelialprogenitor cells (EPCs) in periphal blood of Fgfr1^-/-and Fgfr1fl/flmice was determined by flow cytometry.3. SDF-1αmRNA expression in bone tissue was measured by RT-PCR and SDF-1αlevels in serum, bone marrow extracellular fluid and bone tissue were measured by ELISAassay.4. Primary calvarial osteoblasts from wild-type and FGFR1mutant mice were culturedand treated with LPS, and the expression of SDF-1α mRNA and secretion of SDF-1α proteinof these LPS-treated cells were determined.ResultsPart Ⅰ Serum level of Bone Alkaline Phosphatase can be used to assess illnessseverity of infected neonates in the Neonatal Intensive Care Unit1. The serum BALP levels were lower in the infected group than that in the non-infectedgroup.2. Serum BALP levels were increased markedly in the infected neonates after treatment.3. The serum BALP levels were inversely correlated with SNAPPE of infected neonatesbefore and after treatment. In infected neonates, the differences in serum BALP levels beforeand after treatment were inversely correlated with the IL-6level.4. There were no significant changes in the OC, CTX and PCT levels in the infected ornon-infected groups, before and after treatment.Part Ⅱ Abnormal bone resulting from osteoblast-specific deletion of FGFR1affectsthe outcome of septic mice by mediating mobilization of EPC into peripheral blood viaits regulation of SDF-1α level1. Fgfr1^-/-mice showed a longer survival time than that of Fgfr1fl/flmice after CLP.2. EPCs number and SDF-1α mRNA expression in bone tissue were higher in Fgfr1^-/-mice than those in Fgfr1fl/flmice after injection of LPS.3. SDF-1a level was increased in serum and bone tissues, but decreased in bone marrowof Fgfr1^-/-mice.4. Primary Fgfr1^-/-osteoblasts treated with LPS showed higher levels of SDF-1α mRNAand secreted SDF-1α protein.Conclusions:1. Serum BALP might be used as a marker for assessing the severity of illness ininfected neonates. 2. Osteoblast-specific knock out of FGFR1in mice can improve their survival fromsepsis by mobilizng EPCs into periphal blood through its up-regulation of SDF-1α expressionin OBs.