Targeting the Plant Inside

Toxoplasm gondii are intracellular parasites that cause toxoplasmosis, the “third leading cause of death attributed to foodborne illness in the United States. More that 60 million [Americans] carry the Toxoplasma parasite, but very few have symptoms because the immune system usually keeps the parasite from causing illness” (CDC). T.gondii contain organelles or plastids called “apicoplasts” that were probably acquired in evolution along with an algal endosymbiont.

Apicoplasts contain unique proteins that may be effectively targeted by therapeutic agents. These researchers previously demonstrated a calcium control of protein secretion and identified conserved Ca-responsive proteins. Here they show that calcium-dependent development in T. gondii is controlled by a plant hormone produced by the apicoplast, abscisic acid (ABA, named for its role in abscission, the shedding of plant leaves, fruit, etc.). They found that fluridone, an herbicide that specifically inhibits an enzyme on the synthetic pathway producing ABA, blocked T gondii maturation. Finally, they demonstrated that the herbicide also worked as a therapeutic, protecting mice from a lethal dose of T gondii (shown, from Figure 4). Another infamous apicoplast-containing parasite is Plasmodium falciparum, which causes severe malaria, suggesting an exciting new approach to treating this scourge.

Nagamune et al. "Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii" Nature. 2008 Jan 10;451(7175):207-10.

Protracted IKKb inhibition is dangerous

Many pro-inflammatory molecules share the intracellular signaling pathway with Nuclear Factor – kappa Binding (NF-kB) for their induction and activity. Cytoplasmic NF-kB is released and transported into the nucleus, where it drives transcription of specific genes, when an inhibitor (IkB) is phosphorylated by the inhibitor-kappa kinase beta (IKKb) The importance of this pathway suggests that IKKb would be a promising target for new anti-inflammatory drugs. Here, Greten and colleages tested this idea using mice in which IKKb was deleted in myeloid cells: macrophages and neutrophils (IKKb-del-myo). [A total IKKb deficiency is an embryonic lethal]. IKKb-del-myo mice were viable and had no obvious changes even within tissues that are rich in myeloid cells, such as lymphoid organs and the gastrointestinal tract. They observed that IKKb-del-myo mice were actually more susceptible to endotoxin- (LPS-) induced toxic shock, which is mediated by TNF and interleukin-1beta (IL-1b). IKKb-deficient cells are more susceptible to apoptosis because some NF-kB-induced genes are protective (e.g., A20).

The authors also studied another conditional knockout mouse, in which IKKb is deleted in all “interferon-inducible” cells (IKKb-del), though this model is complicated by a requisite induction with the pleiotropic agent poly-I:C and deletion probably occurs in many more cell types. These mice develop many more granulocytes and, like the IKKb-del-myo mice, also produced much higher levels of IL-1b after LPS treatment. However, mRNA encoding IL-1 was actually decreased, suggesting a change in IL-1 processing.

Indeed, the authors determined that NF-kB inhibits caspase-1 and serine proteases that are required in macrophages and neutrophils, respectively, to cleave pro-IL-1b and release the active cytokine. Consistent with this interpretation, a serine protease inhibitor protected mice against IL-1b-induced death. Mice exposed to LPS die more quickly and more often when fed ML120B, an inhibitor of IKKb for a protracted period with multiple doses, correlating with an increased processing and release of IL-1b (figure, extracted from Figure 7). Definitely not a lead compound for a daily regimen.
Greten et al. Cell. 2007 Sep 7;130(5):918-31. "NF-kappaB is a negative regulator of IL-1beta secretion as revealed by genetic and pharmacological inhibition of IKKbeta"

Fatter and Sicker: Obesity weakens Immunity

This paper reminds me of an old joke: when a patient, having received a dubious diagnosis, asks for a second opinion, the doctor replies 'you're also ugly'. But seriously, folks, this paper suggests that the rise of obesity in the US could have unanticipated consequences for infectious disease. To test the effect of obesity on immunity, Amar and colleagues infected fat or lean mice with Porphyromonas gingivalis, a bacterium that causes gum disease. Mice fed with high fat food for 4 months weighed about 30% more than the mice on normal chow.
They found that mice with diet-induced obesity (DIO) suffered a greater loss of alveolar (gum) bone than did lean mice. P. gingivalis infection induced less inflammatory cytokine release in DIO mice than in lean mice. Macrophages cultured from DIO mice showed significantly reduced activation by LPS of the intracellular signaling molecule NF-kappaB (a chromosome immunoprecipitation is shown, from Fig. 8). Oddly, C-reactive protein, a marker of inflammation, is reported elevated in the bloodstream of obese humans. Do you have a 'second opinion'?
Amar et al., Diet-induced obesity in mice causes changes in immune responses and bone loss manifested by bacterial challenge. Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20466-71.

T cell diversity

Patrick writes: "Virginal CD8 T cells encountering MHC-associated antigen will undergo activation, proliferation, gaining effector functions, followed by contraction and the development of central and effector T cell memories. A number of models have been proposed to account for the generation of these T cell subsets. The observation of homeostatic proliferation suggests that virginal CD8 T cells may not have been as naïve as one have previously thought. This proliferation may have given rise to intraclonal diversity and pre-programmed the “naïve” precursors to develop into differing subsets when encountering antigens. An alternative “progressive differentiation” model proposes that different signal strength at the time of priming may give rise to the diverse subsets. An extension of this model is that a stronger signal may impact the differentiation pattern of one naïve T cells encountering antigen on a DC in contrast to two or three naïve T cells encountering the antigen on the same DC. A “latecomer effect”, i.e., a naïve T cell encountering the antigen late, also may drive the development of long-living T cells.

In this report (Immunity. 2007 Dec 21;27(6):985-997), Stemberger et al. developed a single cell adoptive transfer system to show one CD8+ CD45+ naïve T cell can give rise to diverse phenotypic subsets with functionality (as measured by degranulation, IL-2, IFNg and TNFα expression), including T effectors (CD127-lo, CD62L-lo); effector memory T cells (CD127-hi, CD62L-lo); and, central memory T cells (CD127-hi, CD62L-hi). The observed phenotypic and functional patterns of these subsets are comparable to those seen in resident CD8+ CD45+ T cells upon antigen activation. This in vivo assay, while technically challenging, may provide a handle to study the factors that promote the differentiation and development of effector and central memories."
Stemberger et al. Immunity. 2007 Dec 21;27(6):985-997. A Single Naive CD8(+) T Cell Precursor Can Develop into Diverse Effector and Memory Subsets.

T cell diversity: E Unum Pluribus

Infections stimulate the expansion of a few individual progenitor T lymphocytes, perhaps as few as 50, into millions of specific T lymphocytes (Review). These T cell clones are typically diverse, encompassing subsets of short-lived effector cells, long-lived memory cells, and a variety of intermediate subsets. The proposed models of diversification focus on the priming phase because previous observations have shown that an initial, transient encounter with antigen suffices to induce protracted proliferation and complete subset diversity. Differentiation capabilities might be predetermined, with the naive precursor cells programmed to undergo diversification into all subsets. Alternatively, a ‘‘progressive differentiation’’ model proposes that the strength of antigen stimulation and costimulation at priming determines how far the T cell progeny are driven along a differentiation spectrum.

Here, the ability of precursor T cells to differentiate into multiple subsets was tested by transferring a single naive T cell and then analyzing its progeny after immunization. Twelve days after transfer and immunization of the host with intracellular bacteria expressing ovalbumin, (L.m.-ova), the donor (CD45.1+) transgenic, ova-specific T cells were found to generate many subsets of CD8+ T cells, including CD127+ memory T cells detected in spleen and lymph nodes. The figure, derived from fig. 1A, shows 2 host mice, one on each row; progeny of the transferred cell, the CD45.1+ population circled in the left panels, could be detected in about a quarter of the host mice. Progeny T cells were also detected in lungs. Both 'effector' and 'central' memory T cells (low vs. high CD62L) were detected, the former in the lymphoid organs and latter in the lungs. Upon restimulation of the progeny in vitro, production of the inflammatory cytokines interferon-gamma and tumor-necrosis-factor was detected, demonstrating maturation into effector T cells. This is a straightforward test, albeit technically demanding, that provides strong evidence that a single naïve T cell can generate many, and perhaps all, subsets.
Stemberger et al. Immunity. 2007 Dec 21;27(6):985-997. A Single Naive CD8(+) T Cell Precursor Can Develop into Diverse Effector and Memory Subsets.

How bacteria avoid dying alone

Programmed cell death, PCD or apoptosis, was discovered as the way individual cells altruistically remove themselves during development of a multi-cellular organism (e.g., C. elegans). However, single celled bacteria also undergo PCD to prevent the spread of a bacteriophage infection, for example. Stressed E. coli produce the stable endoribonuclease toxin MazF as well as the labile antitoxin MazE; even a transient interruption of protein synthesis can lead to PCD.

This group previously suggested that mazEF-mediated PCD depends on cell density. Here they show that indeed the mazEF system is only effective above ~3 million cells/ml. Adding supernatant from a dense culture to a diluted culture along with a stress-inducing antibiotic quickly induced PCD in wild-type (WT) cells but not cells in which mazEF is deleted (del-mazEF). This observation led them to identify an “extracellular death factor” (EDF) that is produced during log-phase growth but not during the stationary phase. Here they show that EDF is a pentapeptide (NNWNN) and that adding synthetic EDF to supernatants from stationary phase cultures renders them capable of inducing mazEF-induced PCD (Figure). High EDF (> 200 ng/ml) reduced viability of even del-mazEF strains, which the authors ascribe to perhaps inducing other PCD systems or inactivating essential components. Using mutagenesis, they demonstrated that NNWNN is the optimal sequence for EDF activity. No E. coli genes encode NNWNN but zwf-encoded NNWDN could be amidated to yield NNWNN and deletion of both zwf and ygeO, which encodes a similar NNWN peptide, prevented EDF induction. The authors propose a quorum-sensing role for EDF and point out that synthetic EDF “may be a lead for a new class of antibiotics that specifically trigger bacterial cell death".
Kolodkin-Gal et al. Science. 2007 Oct 26;318(5850):652-5. “A linear pentapeptide is a quorum-sensing factor required for mazEF-mediated cell death in Escherichia coli”.

2007 Flu vaccine: Pain, and gain?

The influenza vaccine contains three influenza virus strains – A (H1N1), A (H3N2), and B. The effectiveness of the vaccine depends on the match between the strains in the vaccine and those that are circulating in the community.

In years when vaccine and circulating strains were poorly matched, the vaccine can be ineffective (Bridges 2000). (Surprisingly, this study also concluded that even in a year when they were well matched, the cost outweighed benefit for people under 65.) In contrast, other studies have demonstrated some benefit even when the vaccine and circulating strains were not well matched. During the 2003-04 season, for example, vaccine effectiveness among people 50-64 years old was over 50% for protecting against contracting flu and 90% against hospitalization (Herrera 2006). The 2003-04 vaccine was similarly effective in children (Ritzwoller 2005). Children are responsible for the most transmission yet they are vaccinated at a lower rate than older adults.

For the current flu season, the Advisory Committee on Immunization Practices (ACIP) recommended vaccination with the strains A/Solomon Islands/3/2006 (H1N1)-like, A/Wisconsin/67/2005 (H3N2)-like, and B/Malaysia/2506/2004. The first detailed analysis of the current flu season was posted last week by the CDC. Between September 30 and December 1, 2007, World Health Organization and National Respiratory and Enteric Virus Surveillance System tested 24,897 respiratory specimens for influenza viruses; 559 (2.2%) were positive. Of these, 92% were influenza A viruses, and 8% were influenza B viruses. One hundred thirty-five of the influenza A viruses were subtyped; 83% of these were influenza A (H1) viruses, and 17% were influenza A (H3) viruses. CDC characterized 27 isolates to date (see table): 19 were influenza A (H1) isolates, 5 were influenza A (H3) isolates, and 3 were influenza B isolates. All 19 A/H1 viruses were Solomon Islands/3/2006-like. Two A/H3 isolates were Wisconsin/67/2005-like. Three A/H3 isolates were similar to Brisbane/10/2007, a strain recommended in the vaccines for the Southern Hemisphere. The three influenza B viruses characterized all belong to the Yamagata/16/88 lineage, whereas the Malaysia strain in the vaccine belongs to the Victoria lineage.

The fact that all H1 isolates are in the vaccine indicates that ACIP accurately predicted the rise of this strain. Although the efficacy of the vaccine is not established, its good match with the circulating strains suggests it will be beneficial. However, the H3 component matches only a minority of the circulating strains and the B component match none. Fortunately, these strains are less prevalent than the H1 strains.

Reap before sowing: depleting host Hematopoetic Stem Cells improves transplantation

Hematopoetic stem cells (HSC) can generate all the cells of the blood, including the myeloid (erythrocytes, neutrophils, etc.) and lymphoid (B, T, and NK cells) lineages. HSC transplantation would be a valuable therapy for many conditions, including reconstitution of immune deficiencies and following radiation.

HSCs transplanted by intravenous injection efficiently home to the bone marrow. However, transplanted HSCs are only transiently productive, with donor cell frequencies in the blood reduced to < 1% within months, unless the host is “conditioned” by toxic regimens that are thought to work by depleting host HSC occupying a limiting number of niches in the bone marrow.

Here, Czechowicz and colleagues tested a targeted depletion with ACK2, an antibody that blocks the cytokine stem cell factor (SCF) receptor CD117 (c-kit). They report that ACK2 treatment of immunodeficient mice led to the transient removal of >98% of HSCs. (Using immunodeficient hosts avoided host-vs.-graft immune responses that would complicate the interpretation. Rag2-knockout mice lack mature B and T cell lineages due to failure to recombine V(D)J regions of immunoglobulin and T cell receptor genes. Common-gamma-chain-knockout mice are severely immunodeficient due to the failure to signal IL2, IL4, IL7, IL9, and IL15. ) Moreover, they show that donor HSC in ACK2-treated immunodeficient host mice produced up to 90% of the blood cells months after transplantation (figure 3a). This result demonstrates that host HSC must be depleted from a limiting number of niches to allow donor HSC to stably reconstitute an immunodeficent host.

Czechowicz et al. "Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches". Science. 2007 Nov 23;318(5854):1296-9.

T(H)-17 & Pathology

A colleague writes: "The recent group of papers in Nature Immunology (Stumhofer, Awasthi, and Fitzgerald) concerning TH-17 cells is driven, at least in part, by a question as old as the finding that some T cell lines or clones can cause a model inflammatory disease, EAE: What distinguishes pathogenic T cells in an EAE model from those cells that cannot cause inflammatory disease? With the emergence of the TH1-TH2 paradigm, it became “clear” that the pathogenic cells were of the proinflammatory, TH1 persuasion. This belief was then shaken by experiments using antibodies to, and knockouts of, to the p35 and p40 subunits of IL-12, which revealed that the “key cytokine” leading to pathogenicity was not the TH1-promoting IL-12 itself, but rather IL-23, which shares the p40 subunit. IL-23 was shown to promote a T cell population that produced TNF, IL-6, and IL-17, among other factors. Further study showed that differentiation of T cells toward this new “TH-17” phenotype was most effectively driven by IL-6 in combination with TGF-beta, whereas TGF-beta alone drove expression of Foxp3 and emergence of regulatory T cells.

This new group of papers incorporates a variety of approaches that all lead toward similar conclusions: TH-17s do not constitute a monolithic proinflammatory population, but can be influenced by either IL-27 or the combination of TGF-beta and IL-6 to include cells that secrete IL-10 in addition to IL-17 and that thus suppress inflammatory disease. As the figure from the paper by McGeachy et al. shows, IL-23 and the combination of TGF-beta and IL-6 both induce secretion of IL-17 by T cells during a recall response, but only the IL-23-treated cells cause EAE when transferred into naïve hosts. Other results in this paper and others show that this protection from disease is attributable to IL-10, which is secreted by T cells in response to IL-27 or the TGF-beta/IL-6 combination. These results may point the way to new modalities for steering otherwise harmful autoimmune responses into more benign channels."

McGeachy et al. "TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology." Nat Immunol. 2007 Dec;8(12):1390-7.

T cell maintenance: Care and Feeding

An abstract with the words ‘dynamic’ and ‘interplay’ usually signals a paper to be avoided but this one tells an interesting story about T cell survival. T cells require occasional stimulation through their antigen receptor (TCR) and feeding with interleukin-7 (IL-7), a product of stromal cells, monocytes, and some epithelial cells. T cells seem to share a limited amount of IL-7 through a negative feedback on the receptor gene transcription (IL-7Ra).

Tickling the TCR is trickier: too much could provoke autoimmunity, too little and the T cell dies. Here, Park and colleagues show that IL-7 (and other gamma common chain agonists) also regulates transcription of the CD8 gene encoding the monomorphic co-receptor of the TCR on a large subset of T cells. Moreover, they report that TCR signals from endogenous antigens inhibit CD8 expression, thereby promoting self tolerance.
The balance is illustrated in HY RAG mice, which are are engineered to contain only T cells specific for HY (a tissue antigen encoded on the Y-chromosome and therefore expressed only in males). If no antigen is present (females), then there is no TCR engagement, leading to more CD8 expression and less IL-7Ra expression than normal (Figure, top vs. second line). With more (and more) TCR engagement in males with appropriate H-2b alleles, comes less CD8 and more IL-7Ra expression (bottom 2 lines). They term this dynamic interplay “coreceptor tuning”.
Park JH, et al. Nat Immunol. 2007 Oct;8(10):1049-59. 'Coreceptor tuning': cytokine signals transcriptionally tailor CD8 coreceptor expression to the self-specificity of the TCR.