MyD88 adapter-like (Mal)/TIRAP interaction with TRAF6 is critical for TLR2- and TLR4-mediated NF-kappaB proinflammatory responses

Toll/interleukin-1 (TIR)receptor-containing adapters are critical in orchestrating the different signal transduction pathways following Toll-like receptor (TLR) activation. MyD88 adapter-like (Mal), also termed TIRAP, is involved in bridging MyD88 to the receptor complex for TLR-2 and TLR4 signaling in response to bacterial infection. We have previously reported an interaction between Mal and tumor necrosis factor receptor-associated factor 6 (TRAF6) via a TRAF6-binding motif, the disruption of which inhibited TLR-mediated NF-kappaB-luciferase reporter activity. Given the recent report of intracellular TRAM localization promoting sequential signaling in TLR4 responses, we further characterized Mal interaction with TRAF6, the cellular localization, and the outcomes of disrupting this association on TLR inflammatory responses. We found that Mal and TRAF6 directly interact in response to TLR2 and TLR4 stimulation, although membrane localization is not necessary to facilitate interaction. Critically, reconstitution of murine Mal-deficient macrophages with MalE190A, containing a mutation within the TRAF6-binding motif, fails to reconstitute the proinflammatory response to TLR2 and TLR4 ligands compared with wild type Mal. Furthermore, Mal interaction with TRAF6 mediates Ser phosphorylation of the p65 subunit of NF-kappaB and thus controls transcriptional activation but not nuclear translocation of NF-kappaB. This study characterizes the novel role for Mal in facilitating the direct recruitment of TRAF6 to the plasma membrane, which is necessary for TLR2- and TLR4-induced transactivation of NF-kappaB and regulation of the subsequent pro-inflammatory response

Increased survival in B-cell-deficient mice during experimental cerebral malaria suggests a role for circulating immune complexes

The pathogenesis of malaria, an insect-borne disease that takes millions of lives every year, is still not fully understood. Complement receptor 1 (CR1) has been described as a receptor for Plasmodium falciparum, which causes cerebral malaria in humans. We investigated the role of CR1 in an experimental model of cerebral malaria. Transgenic mice expressing human CR1 (hCR1(+)) on erythrocytes were infected with Plasmodium berghei ANKA and developed cerebral malaria. No difference in survival was observed in hCR1(+) mice compared to wild-type mice following infection with P. berghei ANKA; however, hCR1 detection was significantly diminished on erythrocytes between days 7 and 10 postinfection. hCR1 levels returned to baseline by day 17 postinfection in surviving animals. Immunoblot assays revealed that total erythrocyte hCR1 levels were diminished, confirming that immune complexes in association with erythrocyte hCR1 were likely removed from erythrocytes in vivo by clearance following immune adherence. Decreases in hCR1 were completely dependent on C3 expression, as mice treated with cobra venom factor (which consumes and depletes C3) retained hCR1 on erythrocytes during C3 depletion through day 7; erythrocyte hCR1 decreases were observed only when C3 levels recovered on day 9. B-cell-deficient mice exhibit a marked increase in survival following infection with P. berghei ANKA, which suggests that immune complexes play a central role in the pathogenesis of experimental cerebral malaria. Together, our findings highlight the importance of complement and immune complexes in experimental cerebral malaria. IMPORTANCE Cerebral malaria is a deadly complication of infection with Plasmodium falciparum. Despite its high prevalence, relatively little is understood about its pathogenesis. We have determined that immune complexes are generated and deposited on erythrocytes specifically expressing human complement receptor 1 in a mouse model of cerebral malaria. We also provide evidence demonstrating the importance of immunoglobulins in the pathogenesis of cerebral malaria in mice. These findings may have important implications in human cerebral malaria

DOCK8 Functions as an Adaptor that Links TLR–MyD88 Signaling to B Cell Activation

DOCK8 and MyD88 have been implicated in serologic memory. Here we report antibody responses were impaired and $CD27^+$ memory B cells were severely reduced in DOCK8-deficient patients. Toll-like receptor 9 (TLR9)- but not CD40-driven B cell proliferation and immunoglobulin production were severely reduced in DOCK8-deficient B cells. In contrast, TLR9-driven expression of AICDA, CD23 and CD86, and activation of NF-κB, p38 and Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. Following TLR9 ligation, DOCK8 became tyrosine phosphorylated by Pyk2, bound the Src family kinase Lyn and linked TLR9 to a Src-Syk-STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells

MD-2 is required for disulfide HMGB1-dependent TLR4 signaling

Innate immune receptors for pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) orchestrate inflammatory responses to infection and injury. Secreted by activated immune cells or passively released by damaged cells, HMGB1 is subjected to redox modification that distinctly influences its extracellular functions. Previously, it was unknown how the TLR4 signalosome distinguished between HMGB1 isoforms. Here we demonstrate that the extracellular TLR4 adaptor, myeloid differentiation factor 2 (MD-2), binds specifically to the cytokine-inducing disulfide isoform of HMGB1, to the exclusion of other isoforms. Using MD-2–deficient mice, as well as MD-2 silencing in macrophages, we show a requirement for HMGB1-dependent TLR4 signaling. By screening HMGB1 peptide libraries, we identified a tetramer (FSSE, designated P5779) as a specific MD-2 antagonist preventing MD-2–HMGB1 interaction and TLR4 signaling. P5779 does not interfere with lipopolysaccharide-induced cytokine/chemokine production, thus preserving PAMP-mediated TLR4–MD-2 responses. Furthermore, P5779 can protect mice against hepatic ischemia/reperfusion injury, chemical toxicity, and sepsis. These findings reveal a novel mechanism by which innate systems selectively recognize specific HMGB1 isoforms. The results may direct toward strategies aimed at attenuating DAMP-mediated inflammation while preserving antimicrobial immune responsiveness

Neutrophil Paralysis in Plasmodium vivax Malaria

Plasmodium vivax is responsible for approximately 60–80% of the malaria cases in the world, and contributes to significant social and economic instability in the developing countries of Latin America and Asia. The pathogenesis of P. vivax malaria is a consequence of host derived inflammatory mediators. Hence, a better understanding of the mechanisms involved in induction of systemic inflammation during P. vivax malaria is critical for the clinical management and prevention of severe disease. The innate immune receptors recognize Plasmodium sp. and initiate a broad spectrum of host defense mechanisms that mediate resistance to infection. However, the innate immune response is the classic “two-edged sword”, and clinical malaria is associated with high levels of circulating pro-inflammatory cytokines. Our findings show that both monocytes and neutrophils are highly activated during malaria. Monocytes produced high levels of IL-1β, IL-6 and TNF-α during acute malaria. On the other hand, neutrophils were a poor source of cytokines, but displayed an enhanced phagocytic activity and superoxide production. Unexpectedly, we noticed an impaired chemotaxis of neutrophils towards an IL-8 (CXCL8) gradient. We proposed that neutrophil paralysis is in part responsible for the enhanced susceptibility to bacterial infection observed in malaria patients

A Complete Pathway Model for Lipid A Biosynthesis in Escherichia coli.

Lipid A is a highly conserved component of lipopolysaccharide (LPS), itself a major component of the outer membrane of Gram-negative bacteria. Lipid A is essential to cells and elicits a strong immune response from humans and other animals. We developed a quantitative model of the nine enzyme-catalyzed steps of Escherichia coli lipid A biosynthesis, drawing parameters from the experimental literature. This model accounts for biosynthesis regulation, which occurs through regulated degradation of the LpxC and WaaA (also called KdtA) enzymes. The LpxC degradation signal appears to arise from the lipid A disaccharide concentration, which we deduced from prior results, model results, and new LpxK overexpression results. The model agrees reasonably well with many experimental findings, including the lipid A production rate, the behaviors of mutants with defective LpxA enzymes, correlations between LpxC half-lives and cell generation times, and the effects of LpxK overexpression on LpxC concentrations. Its predictions also differ from some experimental results, which suggest modifications to the current understanding of the lipid A pathway, such as the possibility that LpxD can replace LpxA and that there may be metabolic channeling between LpxH and LpxB. The model shows that WaaA regulation may serve to regulate the lipid A production rate when the 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) concentration is low and/or to control the number of KDO residues that get attached to lipid A. Computation of flux control coefficients showed that LpxC is the rate-limiting enzyme if pathway regulation is ignored, but that LpxK is the rate-limiting enzyme if pathway regulation is present, as it is in real cells. Control also shifts to other enzymes if the pathway substrate concentrations are not in excess. Based on these results, we suggest that LpxK may be a much better drug target than LpxC, which has been pursued most often

The Genome of Anopheles darlingi, the main neotropical malaria vector

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vectorhuman and vectorparasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles- darlingi. © 2013 The Author(s)