A Subset of Type I Conventional Dendritic Cells Controls Cutaneous Bacterial Infections through VEGFα-Mediated Recruitment of Neutrophils

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Molecular epidemiology and evolution of mosquito-borne flaviviruses and alphaviruses enzootic in Australia

Three distinct patterns in the molecular epidemiology and evolution are evident among the alphaviruses and flaviviruses enzootic in Australia. One pattern, exemplified by MVE and KUN viruses, is of a single genetic type evolving slowly and uniformly in geographically widely separated regions of Australia with no evidence of independent divergence. The second pattern, exemplified by RR virus, is of separate genotypes evolving in different geographic regions with significant nucleotide divergence between genotypes. The third pattern, exemplified by SIN virus, is of a succession of temporally related genotypes that extend over most of the Australian continent, with relatively low levels of nucleotide divergence within a genotype, and which are each replaced by the subsequent genotype. These patterns are associated in part due to the nature and dispersal of their vertebrate hosts. Nucleotide divergence rates for Australian alphaviruses are similar to those reported elsewhere. Genomic relationships between Australian flavivirus members of the JE virus serological complex and between Australian alphaviruses are discussed, and evidence is presented for a possible new genomic lineage of SIN virus.     

The distinctive germinal center phase of IgE+ B lymphocytes limits their contribution to the classical memory response

The mechanisms involved in the maintenance of memory IgE responses are poorly understood, and the role played by germinal center (GC) IgE⁺ cells in memory responses is particularly unclear. IgE⁺ B cell differentiation is characterized by a transient GC phase, a bias toward the plasma cell (PC) fate, and dependence on sequential switching for the production of high-affinity IgE. We show here that IgE⁺ GC B cells are unfit to undergo the conventional GC differentiation program due to impaired B cell receptor function and increased apoptosis. IgE⁺ GC cells fail to populate the GC light zone and are unable to contribute to the memory and long-lived PC compartments. Furthermore, we demonstrate that direct and sequential switching are linked to distinct B cell differentiation fates: direct switching generates IgE⁺ GC cells, whereas sequential switching gives rise to IgE⁺ PCs. We propose a comprehensive model for the generation and memory of IgE responses.IgE antibodies are critical mediators of allergic reactions (Gould and Sutton, 2008). Cross-linking of IgE molecules bound to high affinity FcεRI receptors on mast cells and basophils leads to the rapid release of potent proinflammatory molecules (Kinet, 1999; Galli and Tsai, 2012). In spite of its pathological potential, IgE exhibits the lowest serum concentration and the shortest half-life of all the antibody isotypes (Vieira and Rajewsky, 1988; Gould and Sutton, 2008). The low frequency of IgE-producing cells makes their study particularly challenging. Using mouse models of high IgE responses (Katona et al., 1988; Curotto de Lafaille et al., 2001), we discovered that IgE-producing cells develop via a unique differentiation pathway that occurs during the germinal center (GC) phase of T cell–dependent responses and yet favors the production of plasma cells (PCs; Erazo et al., 2007; Yang et al., 2012). In our early studies a GC IgE⁺ population was not clearly detectable, but the IgE antibodies produced were observed to have undergone affinity maturation, indicating a GC history for IgE⁺ PC. We proposed at the time that high affinity IgE originated from the sequential switching of high affinity IgG1 cells, and hence we speculated that classical IgE⁺ memory cells may be absent in mice (Erazo et al., 2007; Curotto de Lafaille and Lafaille, 2010).Sequential switching of IgG cells to IgE was first discovered by the identification of switch(S)γ region footprints in the Sμ-Sε DNA region of IgE genes (Matsuoka et al., 1990; Yoshida et al., 1990; Jabara et al., 1993; Mandler et al., 1993; Zhang et al., 1994; Baskin et al., 1997), but the biological significance of this finding was at that time unknown. Sequential switching in mice entails two recombination events, Sμ→Sγ1 and SμSγ1→Sε, that may be either continuous or temporally separate events. The latter scenario allows for the existence of an intermediate IgG1 cellular phase in which affinity maturation can occur in GCs. Indeed, stimulation of IgG1 cells in the presence of IL-4 either in vivo or in vitro resulted in the production of IgE antibodies (Erazo et al., 2007; Wesemann et al., 2012). Importantly, mice deficient in class switching to IgG1 due to a mutation in the Iγ1 exon (Lorenz et al., 1995) were unable to produce high affinity IgE antibodies (Xiong et al., 2012a,b), indicating that sequential switching is essential for the formation of high affinity IgE.The recent development of fluorescent reporter mice for IgE has facilitated the identification of IgE GC cells (Talay et al., 2012; Yang et al., 2012). However, the in vivo phenotype and role of IgE GC cells in supporting IgE responses and its relationship with the sequential switching process remain unclear (Lafaille et al., 2012; Xiong et al., 2012a).In the current study, we used a new reporter mouse for class switch recombination (CSR) to IgE, improved methods to functionally study IgE B cells ex vivo and in vivo, and in silico modeling to analyze the origin, functional properties, and population dynamics of IgE GC cells and PC. We show that IgE GC cells are unfit to undergo the conventional GC differentiation program and instead undergo apoptosis at a high rate. This “failure to thrive” of IgE GC cells greatly limits their contribution to the memory pool and high affinity PC compartment. Furthermore, we show that the two types of rearrangement to IgE are associated with distinct B cell differentiation fates. Direct Sμ-Sε rearrangements generate IgE GC cells, whereas sequential switching of IgG1 cells gives rise to IgE PC.     

Brief report: Decreased expression of CD244 (SLAMF4) on monocytes and platelets in patients with systemic lupus erythematosus

Clinical Rheumatology - The signalling lymphocyte activation molecule (SLAM) family receptors play important roles in modulating immune responses. Previous studies in murine models and patients...     

Effect of a multifaceted educational intervention for anti-infectious measures on sepsis mortality: a cluster randomized trial

Purpose

Guidelines recommend administering antibiotics within 1 h of sepsis recognition but this recommendation remains untested by randomized trials. This trial was set up to investigate whether survival is improved by reducing the time before initiation of antimicrobial therapy by means of a multifaceted intervention in compliance with guideline recommendations.

Methods

The MEDUSA study, a prospective multicenter cluster-randomized trial, was conducted from July 2011 to July 2013 in 40 German hospitals. Hospitals were randomly allocated to receive conventional continuous medical education (CME) measures (control group) or multifaceted interventions including local quality improvement teams, educational outreach, audit, feedback, and reminders. We included 4183 patients with severe sepsis or septic shock in an intention-to-treat analysis comparing the multifaceted intervention (n = 2596) with conventional CME (n = 1587). The primary outcome was 28-day mortality.

Results

The 28-day mortality was 35.1% (883 of 2596 patients) in the intervention group and 26.7% (403 of 1587 patients; p = 0.01) in the control group. The intervention was not a risk factor for mortality, since this difference was present from the beginning of the study and remained unaffected by the intervention. Median time to antimicrobial therapy was 1.5 h (interquartile range 0.1–4.9 h) in the intervention group and 2.0 h (0.4–5.9 h; p = 0.41) in the control group. The risk of death increased by 2% per hour delay of antimicrobial therapy and 1% per hour delay of source control, independent of group assignment.

Conclusions

Delay in antimicrobial therapy and source control was associated with increased mortality but the multifaceted approach was unable to change time to antimicrobial therapy in this setting and did not affect survival.

     

RNA sensing by conventional dendritic cells is central to the development of lupus nephritis

Glomerulonephritis is a common and debilitating feature of systemic lupus erythematosus (SLE). The precise immune mechanisms that drive the progression from benign autoimmunity to glomerulonephritis are largely unknown. Previous investigations have shown that a moderate increase of the innate Toll-like receptor 7 (TLR7) is sufficient for the development of nephritis. In these systems normalization of B-cell TLR7 expression or temporal depletion of plasmacytoid dendritic cells (pDCs) slow progression; however, the critical cell that is responsible for driving full immunopathology remains unidentified. In this investigation we have shown that conventional DC expression of TLR7 is essential for severe autoimmunity in the Sle1Tg7 model of SLE. We show that a novel expanding CD11b⁺ conventional DC subpopulation dominates the infiltrating renal inflammatory milieu, localizing to the glomeruli. Moreover, exposure of human myeloid DCs to IFN-α or Flu increases TLR7 expression, suggesting they may have a role in self-RNA recognition pathways in clinical disease. To our knowledge, this study is the first to highlight the importance of conventional DC-TLR7 expression for kidney pathogenesis in a murine model of SLE.The innate pathogen recognition receptor Toll-like receptor 7 (TLR7) has recently been implicated in the development of autoimmunity. This receptor, along with TLR8 and TLR9, was originally described by Beutler and colleagues (1), with ligand identification and functional characterization by Akira and colleagues just over a year later (2). TLR7 recognizes single-stranded RNA (ssRNA), inducing downstream activation of signaling molecules, including Jnk and NF-κB through a myeloid differentiation primary response gene 88 (MyD88)-dependent cascade (3). This process is central to host defense against invading viruses; however, TLR7 hyperactivity can also drive the initiation and progression of autoimmunity.Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by the presence of antinuclear autoantibodies (ANA) (4). The ANAs form immune complexes with host-derived nuclear material, which accumulate as deposits within the tissues and organs. The ensuing inflammation can lead to organ failure, particularly glomerulonephritis (GN) (5). Prospective studies by Harley and colleagues (6) demonstrated that ANAs are detectable ∼6 years before clinical presentation of SLE, with only a small percentage of these individuals who have ANAs progressing to pathogenic autoimmunity. These data, together with murine studies, suggest that defects in multiple pathways contribute to the initiation and progression of systemic autoimmunity (7).Multiple investigations have shown that TLR7 and the MyD88 signaling pathway are critical for the initiation of autoimmunity and development of self-reactivity, because genetic ablation of either TLR7 or MyD88 prevents the development of both ANAs and subsequent immune-pathology (8–10). Furthermore, this signaling pathway is specifically required within B cells (11, 12). In TLR7-sufficient autoimmune prone systems, additional immune alterations, such as lpr (lymphoproliferation), sle3, or yaa (Y-linked autoimmune-accelerating locus) can lead to severe autoimmunity. The yaa murine susceptibility locus is a region comprising 16 genes, including TLR7, which translocated from the X chromosome to the Y (13, 14). The yaa-associated increase in TLR7 expression and function was determined to be critical for the development of severe disease, independent of the SLE-model (15–17). More recently, we demonstrated that a twofold increase in TLR7 alone on the Sle1 background is sufficient to drive disease in an almost identical manner to the addition of the yaa susceptibility locus (18). Normalization of B-cell TLR7 did not affect GN, suggesting that the increase in other cells drives severe pathology. Additional studies have suggested that although plasmacytoid dendritic cells (pDCs) contribute to pathology of a yaa-associated model, other cell types are critical for full disease pathogenesis (19). Determining these critical cell types will clarify the mechanisms of progression for therapeutic targeting.In this study we investigated the contribution of DCs to TLR7-mediated SLE-associated GN. Previous data has presented conflicting evidence on their role in lupus models. Although B-cell proliferation and antibody production can be enhanced by DC activation and genetic ablation diminishes disease progression (20, 21), DCs also play a protective role in regulatory T-cell development and their elimination can result in autoimmunity (22, 23). The overall response in each system may depend on the genetic background or incomplete ablation of DC subsets [discussed by Platt and Randolph (24)].We bred conditional SLE mice overexpressing TLR7 (Sle1Tg7) with a CD11c^Cre reporter strain (25) and demonstrated that the increase in TLR7 within CD11c⁺ cells was essential for all severe autoimmune traits, including splenomegaly, T- and B-cell activation, and GN. Purification of DCs and other leukocyte subsets confirmed normalization of TLR7 mRNA in CD11b⁺ conventional (c)DCs and pDCs. We identified and characterized a novel CD11b⁺ cDC infiltrating diseased kidneys, which was absent, together with all other pathological traits, upon restoration of TLR7. Taken together, our data suggest that the CD11b⁺ cDC, rather than the pDC, is playing the principle role in driving end organ disease. Finally, we determined that primary human BDCA1⁺ DCs or monocyte-derived DCs express extremely low levels of TLR7 mRNA, but this can be rapidly up-regulated on exposure to virus or IFN-α. This work has uncovered a key role for DCs in one of the most clinically important phases of SLE, the progression to lupus nephritis.     

The molecular epidemiology of Kokobera virus

We describe herein the molecular epidemiology and phylogeny of Kokobera (KOK) virus, a flavivirus found in Australia and Papua New Guinea. We sequenced a region encompassing the 200 nucleotides of the 3' terminus of the NS5 gene, and the first 300 nucleotides of the 3' untranslated region (UTR). The study included 25 isolates of the virus, including an isolate from PNG, and several recent isolates from the south-west of Western Australia (WA), where the virus had not previously been detected. We found that the KOK isolates clustered according to geographic location and time of isolation into three distinct topotypes: one covering Queensland and New South Wales; another represented by the single isolate from PNG; and a third covering the Northern Territory and WA. This latter group was further subdivided into northern and south-west isolates. This molecular epidemiology is significantly different from other Australian flaviviruses, such as Murray Valley encephalitis (MVE) and Kunjin (KUN) viruses, which exist as single genetic types across the entire Australian continent. However, it is similar to the molecular epidemiology of the alphavirus Ross River (RR) virus. This may be explained by the fact that MVE and KUN viruses are known to have birds as their main vertebrate hosts, whereas RR virus utilises macropods, which have also been implicated as the vertebrate host for KOK virus. In addition, the south-west isolates exhibited a degree of sequence heterogeneity, including one isolate that has a nine nucleotide deletion in the 3'UTR. This suggests that KOK virus has been in the south-west of WA for some time, and was not recently introduced.     

Protumoral role of monocytes in human B-cell precursor acute lymphoblastic leukemia: involvement of the chemokine CXCL10

Myelomonocytic cells play a key role in the progression of many solid tumors. However, very little is known about their contribution to the progression of hematopoietic cancers. We investigated the role of monocytes in the progression of human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We demonstrated that coculturing human monocytes in vitro with CD19+ BCP-ALL blasts from patients "conditioned" them to an inflammatory phenotype characterized by significant up-regulation of the chemokine, CXCL10. This phenotype was also observable ex vivo in monocytes isolated from BCP-ALL patients, which show elevated CXCL10 production compared with monocytes from healthy donors. Functionally, the "conditioned" monocytes promoted migration and invasive capacity of BCP-ALL cells. Increased invasion was mediated by matrix metalloproteinase 9 expression and activity in the BCP-ALL cells induced by the monocyte-derived CXCL10. However, neither the "conditioned" monocytes nor the CXCL10 produced by these cells had any effect on the proliferation/viability of BCP-ALL cells and angiogenesis. Collectively, our results strongly suggest a protumoral role for human monocytes in BCP-ALL, orchestrated by CXCL10 and its effect on tumor cell migration and invasion. These observations highlight the importance of the CXCL10/CXCR3 chemokine circuit in BCP-ALL progression.