Fibroblast growth factor 23 (FGF23) production is upregulated by iron deficiency and hypoxia. However, the influence of acute blood loss, and the resulting increases in circulating erythropoietin, on FGF23 production is unknown. Using wild-type C57BL/6 mice, we show that acute loss of 10% total blood volume leads to an increase in plasma C-terminal FGF23 (cFGF23) levels within 6 h, while plasma levels of intact FGF23, phosphate, calcium, parathyroid hormone, iron, and ferritin remain similar to control mice without acute blood loss. Volume resuscitation with PBS did not significantly alter these findings. The increase in plasma cFGF23 levels in bled animals was accompanied by increased plasma erythropoietin levels at 6 h. Administration of erythropoietin led to an acute increase in plasma cFGF23 levels similar to that observed in acute blood loss. Fgf23 mRNA expression was increased 20-fold in bone marrow, but not in bone, of bled vs. control mice, suggesting bone marrow as a key source of elevated plasma FGF23 levels following acute blood loss. To extend these findings to humans, we measured plasma cFGF23 levels in 131 critically ill patients admitted to the intensive care unit. In univariate and multivariate models, we found a positive association between number of red blood cell transfusions, an indirect indicator of acute blood loss, and plasma cFGF23 levels. We conclude that FGF23 production is rapidly increased after acute blood loss and that erythropoietin may be the mediator of this increase. Thus erythropoietin may represent a novel physiological regulator of FGF23 production.

Background:The mechanism of repression of inflammasome caused by Francisella tularensis is not known. Results: F. tularensis represses AIM2 and NLRP3 inflammasomes in a FTL_0325-dependent fashion. Conclusion: Repression of inflammasome by F. tularensis results in fulminate infection.Significance: This study advances the understanding of mechanisms of immune suppression caused by F. tularensis.

We aimed to investigate the potential relationship between alarmins [acting via Toll-like receptor-4 (TLR4)], uric acid (UA), and high-mobility group box-1 protein (HMGB1) during acute kidney injury. UA, which is significantly increased in the circulation following renal ischemia-reperfusion injury (IRI), was used both in vitro and in vivo as an early response-signaling molecule to determine its ability to induce the secretion of HMGB1 from endothelial cells. Treatment of human umbilical vein endothelial cells (HUVEC) with UA resulted in increased HMGB1 mRNA expression, acetylation of nuclear HMGB1, and its subsequent nuclear-cytoplasmic translocation and release into the circulation, as determined by Western blotting and immunofluorescence. Treatment of HUVEC with UA and a calcium mobilization inhibitor (TMB-8) or a MEK/Erk pathway inhibitor (U0126) prevented translocation of HMGB1 from the nucleus, resulting in reduced cytoplasmic and circulating levels of HMGB1. Once released, HMGB1 in autocrine fashion promoted further HMGB1 release while also stimulating NF-κB activity and increased angiopoietin-2 expression and protein release. Transfection of HUVEC with TLR4 small interfering (si) RNA reduced HMGB1 levels during UA and HMGB1 treatment. In summary, UA after IRI mediates the acetylation and release of HMGB1 from endothelial cells by mechanisms that involve calcium mobilization, the MEK/Erk pathway, and activation of TLR4. Once released, HMGB1 promotes its own further cellular release while acting as an autocrine and paracrine to activate both proinflammatory and proreparative mediators.

Background:The mechanism of immune suppression caused by Francisella tularensis SchuS4 strain, a category A agent, are yet unknown. Results: FTL_0325/FTT0831c genes of F. tularensis suppress proinflammatory cytokines by preventing activation of NF-B signaling. Conclusion: FTL_0325/FTT0831c of Francisella is a key virulence factor and functions as an immunosuppressant. Significance: Understanding of such pathogenic mechanisms will define vaccine candidates to prevent tularemia acquired naturally or through an act of bioterrorism.

Francisella tularensis is the causative agent of a fatal human disease, tularemia. F. tularensis was used in bioweapon programs in the past and is now classified as a category A select agent owing to its possible use in bioterror attacks. Despite over a century since its discovery, an effective vaccine is yet to be developed. In this study four transposon insertion mutants of F. tularensis live vaccine strain (LVS) in Na/H antiporter (FTL_0304), aromatic amino acid transporter (FTL_0291), outer membrane protein A (OmpA)-like family protein (FTL_0325) and a conserved hypothetical membrane protein gene (FTL_0057) were evaluated for their attenuation and protective efficacy against F. tularensis SchuS4 strain. All four mutants were 100–1000 fold attenuated for virulence in mice than parental F. tularensis. Except for the FTL_0304, single intranasal immunization with the other three mutants provided 100% protection in BALB/c mice against intranasal challenge with virulent F. tularensis SchuS4. Differences in the protective ability of the FTL_0325 and FTL_0304 mutant which failed to provide protection against SchuS4 were investigated further. The results indicated that an early pro-inflammatory response and persistence in host tissues established a protective immunity against F. tularensis SchuS4 in the FTL_0325 immunized mice. No differences were observed in the levels of serum IgG antibodies amongst the two vaccinated groups. Recall response studies demonstrated that splenocytes from the FTL_0325 mutant immunized mice induced significantly higher levels of IFN-γ and IL-17 cytokines than the FTL_0304 immunized counterparts indicating development of an effective memory response. Collectively, this study demonstrates that persistence of the vaccine strain together with its ability to induce an early pro-inflammatory innate immune response and strong memory responses can discriminate between successful and failed vaccinations against tularemia. This study describes a live attenuated vaccine which may prove to be an ideal vaccine candidate for prevention of respiratory tularemia.

Francisella tularensis is a Category A Biodefense agent that causes a fatal human disease known as tularemia. The pathogenicity of F. tularensis depends on its ability to persist inside host immune cells primarily by resisting an attack from host-generated reactive oxygen and nitrogen species (ROS/RNS). Based on the ability of F. tularensis to resist high ROS/RNS levels, we have hypothesized that additional unknown factors act in conjunction with known antioxidant defenses to render ROS resistance. By screening a transposon insertion library of F. tularensis LVS in the presence of hydrogen peroxide, we have identified an oxidant sensitive mutant in putative EmrA1 (FTL_0687) secretion protein. The results demonstrate that the emrA1 mutant is highly sensitive to oxidants and several antimicrobial agents, and exhibits diminished intramacrophage growth that can be restored to wild type F. tularensis LVS levels either by transcomplementation, inhibition of ROS generation, or infection in NADPH oxidase deficient (gp91Phox−/−) macrophages. The emrA1 mutant is attenuated for virulence, which is restored by infection in gp91Phox−/− mice. Further, EmrA1 contributes to oxidative stress resistance by affecting secretion of Francisella antioxidant enzymes SodB and KatG. This study exposes unique links between transporter activity and the antioxidant defense mechanisms of F. tularensis.

Summary Francisella tularensis causes a lethal human disease known as tularemia. As an intracellular pathogen, Francisella survives and replicates in phagocytic cells, such as macrophages. However, to establish an intracellular niche, Francisella must overcome the oxidative stress posed by the reactive oxygen species (ROS) produced by the infected macrophages. OxyR and SoxR/S are two well-characterized transcriptional regulators of oxidative stress responses in several bacterial pathogens. Only the OxyR homolog is present in F. tularensis, while the SoxR homologs are absent. The functional role of OxyR has not been established in F. tularensis. We demonstrate that OxyR regulates oxidative stress responses and provides resistance against ROS, thereby contributing to the survival of the F. tularensis subsp. holarctica live vaccine strain (LVS) in macrophages and epithelial cells and contributing to virulence in mice. Proteomic analysis reveals the differential production of 128 proteins in the oxyR gene deletion mutant, indicating its global regulatory role in the oxidative stress response of F. tularensis. Moreover, OxyR regulates the transcription of the primary antioxidant enzyme genes by binding directly to their putative promoter regions. This study demonstrates that OxyR is an important virulence factor and transcriptional regulator of the oxidative stress response of the F. tularensis LVS.

The acute effects of parathyroid hormone (PTH) on fibroblast growth factor 23 (FGF23) in vivo are not well understood. After a single subcutaneous PTH (1-34) injection (50 nmol/kg) in mice, FGF23 levels were assessed in plasma using assays that measure either intact alone (iFGF23) or intact/C-terminal FGF23 (cFGF23). Furthermore, FGF23 messenger RNA (mRNA) and protein levels were assessed in bone. In addition, we examined the effects of PTH treatment on FGF23 production in vitro using differentiated calvarial osteocyte-like cells. cFGF23 levels increased by three- to fivefold within 2 hours following PTH injection, which returned to baseline by 4 hours. In contrast, iFGF23 levels remained unchanged for the first 2 hours, yet declined to ∼60% by 6 hours and remained suppressed before returning to baseline after 24 hours. Using homozygous mice for an autosomal dominant hypophosphatemic rickets-FGF23 mutation or animals treated with a furin inhibitor, we showed that cFGF23 and iFGF23 levels increased equivalently after PTH injection. These findings are consistent with increased FGF23 production in bone, yet rapid cleavage of the secreted intact protein. Using primary osteocyte-like cell cultures, we showed that PTH increased FGF23 mRNA expression through cyclic adenosine monophosphate/protein kinase A, but not inositol triphosphate/protein kinase C signaling; PTH also increased furin protein levels. In conclusion, PTH injection rapidly increases FGF23 production in bone in vivo and in vitro. However, iFGF23 is rapidly degraded. At later time points through an unidentified mechanism, a sustained decrease in FGF23 production occurs.