Infectious Fatigue

Between 1 and 4 million Americans suffer from Chronic Fatigue Syndrome (CFS), which is a “complicated disorder characterized by extreme fatigue that may worsen with physical or mental activity, but doesn't improve with rest.” (Mayo Clinic). The cause is unknown and there is no known cure. An infectious cause has been suspected and sought for decades without success. The recent discovery of XMRV, new retrovirus found in some prostate cancer patients (but not others) prompted Lombardi and colleagues to test for its involvement with CFS. They performed PCR for XMRV gag, which encodes structural viral proteins, on peripheral blood mononuclear cells from CFS patients in a repository at their Whittemore Peterson Institute. Of 101 CFS samples tested, 86 (67%) were positive in contrast to only 8 out of 218 samples (4%) from healthy donors. Another viral gene, env, was also detected in most CFS patients positive for gag as were the proteins encoded by these genes. Oddly, patient sample WPI-1118 is negative for gag and env (Fig 1) but weakly positive by cytometry (Fig 2 A & D) and clearly positive by Western protein blot (Fig 4 A). Both B and T lymphocytes express XMRV proteins. And, for what it's worth, some CFS patient cells make virus that can productively infect other cells (shown, from Figure 3 B & C, electron micrographs of budding virus particles).

The authors note in their introduction that “patients with CFS often have active β herpesvirus infections, suggesting an underlying immune deficiency”. This increases the odds that XMRV is an opportunistic infection. The authors return to this question in their closing discussion, asking “Is XMRV infection a causal factor in the pathogenesis of CFS or a passenger virus in the immunosuppressed CFS patient population?” Stand by.

Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA. "Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic Fatigue Syndrome". Science. 2009 Oct 8.

Infecting the Protectors (fluNK)

Influenza (flu) is typically transmitted through the air, infecting lung epithelial cells. Natural Killer (NK) cells are lymphocytes that help protect from flu, especially early after infection, by killing infected cells and secreting cytokines and chemokines that inhibit virus replication. Here, Guo and colleagues ask whether NK might themselves be infected by flu. They confirmed that NK express on their surface sialic acids, which serve as flu receptors by binding hemagluttinin (HA). By microscopy, they examined mice infected with PR8, a strain originating from the human flu virus A/PR/8/34 and detected flu virus protein M2 inside of NK cells within the lungs of infected mice. NK possess several activating receptors including NCR1, NKG2D, Ly49D and inhibiting receptors including NKG2A, Ly49A. Flow cytometry demonstrated that PR8 did not modulate these receptors or developmental markers.
However, infection did alter the effector functions of NK cells. Infected NK cells were less capable of killing cells from 3 target lines, cells susceptible because they either express ligands for NK activating receptors or fail to express ligands for NK inhibiting receptors (Figure 5, shown). EL4 cells express a transfected H60, which binds the activating receptor NKG2D; YAC-1 cells express H60 constitutively, and RMA-S cells express very little HLA class I protein, which binds the inhibitory Ly49 receptors. The authors and others had previously shown that the 85 kDa subunit of phosphotidyl inositol 3-kinase (PI3K-p85) was critical for NK effector functions, including killing and production of cytokines/chemokines, and that flu NS1 protein interacted with this kinase. Surprisingly, a PR8 virus with a mutated NS1 protein was even more effective in suppressing NK effector functions, leaving the mechanism of inhibition to be determined.
Guo H, Kumar P, Moran TM, Garcia-Sastre A, Zhou Y, Malarkannan S. “The functional impairment of natural killer cells during influenza virus infection.” Immunol Cell Biol. 2009 Sep 1.

Overreacting to Infection is Dangerous to Your Health

Contributed by DP "Following soon after a similar paper on anthrax pathogenesis, this paper suggests that the downregulation of a lymphocyte surface protein can single-handedly stem the destruction by the Ebola virus. The authors identified mice expressing about half (62%) the "wild type" levels of the leukocyte membrane tyrosine kinase CD45 are protected from the lethal effects of Ebola infections (compare CD45-62% to control 100% in the top panel), with an overall survival rate of 90% and a complete clearance of the virus 10 days after challenge. CD45-62% mice expressing CD45 without phosphatase activity did not survive viral challenge, highlighting the importance of CD45’s enzymatic function.

The proposed mechanism is via constitutively high levels of activated CD8 T cells and IFN-gamma because antibodies that kill CD8 cells or block IFN-gamma render CD45-62% mice susceptible to the virus (fig 4, panels B & C). Can this protection translate to other infectious pathogens? Can this information be used clinically? Should we worry about modulating surface CD45 or just give IFN-gamma to affected individuals? I like the former, if one can come up with inexpensive small molecules, rather than the huge expense for IFN-gamma. These molecules might be used at higher doses for GVHD." And what is the evolutionary value of having wt levels of CD45 if reduced levels are in fact protective?

Panchal RG, Bradfute SB, Peyser BD, Warfield KL, Ruthel G, Lane D, Kenny TA, Anderson AO, Raschke WC, Bavari S. Reduced levels of protein tyrosine phosphatase CD45 protect mice from the lethal effects of Ebola virus infection. Cell Host Microbe. 2009 Aug 20;6(2):162-73.

Th17 cells have issues with commitment

Th17 cells, so named because they secrete the protein hormone interleukin-17 (IL-17), belong to a recently-described subset of helper T cells responsible for regulating helper subsets Th1 (cellular) and Th2 (allergic).

Naïve T cells develop into different T helper subsets depending upon culture conditions. Lee and colleagues used an IL-17F reporter mouse (IL17F promoter driving expression of a surface protein Thy1.1) to test the stability of the Th17 phenotype in serial cultures. First, they caused Th17 to develop from naïve CD4+ T cells (OT-II transgene receptors specific for ovalbumin (ova) + MHC class II) by treating them in culture with TGF-beta, IL-6, anti-IFNgamma, and anti-IL-4 in the presence of IL-12p40-deficient antigen-presenting cells (APC). (Culture systems don't get much more manipulated than that!) Then, the surviving cells were phenotyped or cultured with TGNbeta or with IL-23, which stimulates the development of Th1 cells.

Several other groups had reported that Th17 cells required IL-23 for pathogenic autoimmunity. Here, Lee et al. showed that TGFbeta, but not IL-23, is essential for maintaining Th17 commitment (IL-17F expression). However, even after two cultures under Th17 conditions, IL-12 added to subsequent cultures 3 and 4 drove Th1 development and expression of interferon-gamma (IFNg, on the x-axis of figure 2, shown below).

Moreover, this final conversion by Th17 required Th1 factors STAT4 and T-bet, suggesting that Th17 might constitute a prolonged adolescence for adult Th1 cells.

Immunity 30:92-107. “Late developmental plasticity in the T helper 17 lineage”. Lee YK, Turner H, Maynard CL, Oliver JR, Chen D, Elson CO, Weaver CT.

IRF4 etc Required for Th17 Development

Interleukin-17 (IL-17A) is a proinflammatory protein hormone produced by activated T lymphocytes that binds to a ubiquitous low-affinity receptor IL17RA. A subset of T helper (Th) cells that produce IL17 were dubbed Th17 and shown to be induced by IL23 and suppressed by IFNg or IL4, which support the development of Th1 or Th2 cells, respectively.

Brüstle and colleagues tested the ability of lymphocytes to develop into Th17 cells by treating naïve (CD4+CD62L+) Th in culture for 3 days with anti-CD3 and anti-CD28 to stimulate Th0 differentiation. Some cultures also received (1) IL12 and anti-IL4 to stimulate Th1, or (2) IL4 and anti-IFNgamma to stimulate Th2, or (3) anti-IL4, anti-IFNgamma, TGFbeta, IL6, IL1beta, TNF, and IL23 to stimulate Th17. Cells were then restimulated and analyzed by intracellular staining. They found that IRF4-deficient cells differentiate normally into Th1 cells, contradicting an earlier report by the senior author [they attribute the difference to using specific-pathogen-free mice], but not into Th2 cells, as previously reported. They also found that IRF4-deficient cells did not differentiate into Th17 cells. IRF4 is from a family of transcription factors that are required for the production of interferons alpha and beta as well as innate immunity (TLR) signaling and T helper cell differentiation. Mixing cells from normal 'wild-type' (WT) and deficient mice (IRF4-/- also CD45.2+) demonstrated that the defect was intrinsic to the Th17 cell lineage and not due to suppression (figure 1c shown).
They made several other, interesting observations and pursue the IRF4 pathway, finding that the transcription factor RORgamma, also previously reported to be required for Th17 cells, is largely dependent on IRF4. They also showed that TGFbeta-induced FoxP3, a marker of regulatory T cells, is only weakly suppressed by IL6 in IRF4-deficient cells, suggesting that this alternative differentiation pathway may explain the failure of Th17 to develop.
Brüstle A, Heink S, Huber M, Rosenplänter C, Stadelmann C, Yu P, Arpaia E, Mak TW, Kamradt T, Lohoff M. “The development of inflammatory T(H)-17 cells requires interferon-regulatory factor 4” Nat Immunol. 2007 Sep;8(9):958-66

It’s what inside that counts for TLRs 7 & 9

Toll-like receptors (TLRs) help mammalian immune systems protect against infections by binding to molecular patterns that are characteristic of many dangerous microbes. The location of TLR7 and TLR9 within the cell, not on the cell surface, appears crucial to their distinguishing viral from host nucleic acids and maintaining self-tolerance.

Ewald and colleagues determined how these TLRs ‘traffic’ within macrophages and dendritic cells, which are dedicated to picking up and destroying matter, including viruses. They found that these TLRs, after their synthesis in the endoplasmic reticulum, move to vesicles called endolysosomes where recently-internalized materials are degraded. There, a portion of the TLR within the lumen of the lysosome, the 'ectodomain', is cut and destroyed. Both the full-length and ectodomain-cleaved TLRs can bind nucleic acids but only the proteolysed (cleaved) TLR can activate downstream signaling by recruiting MyD88 (Figure 4e shown, anti-Flag-tagged-TLR binds to and co-precipitates HA-tagged TLR after stimulation with nucleic acid). The authors propose that TLR proteolysis and activation within the endolysosome serves to restrict its exposure to 'self' molecules and thereby reduce the likelihood of triggering autoimmunity.

Ewald SE, Lee BL, Lau L, Wickliffe KE, Shi GP, Chapman HA, Barton GM. "The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor." Nature. 2008 Dec 4;456(7222):658-62. Epub 2008 Sep 28.

Kill your ECTV!

It’s a mystery why mice resist the Ectromelia poxvirus (ECTV) better than humans resist smallpox, a similar infection. Previous studies demonstrated a role for natural killer (NK) cells in ECTV resistance. Fang and colleagues show here that in addition to 'opportunity', actual NK killing with perforin is essential to the resolution. Many T cells armed with IFN-g were also found in the spleens of infected mice, suggesting that NK cells involve T cells in the murderous conspiracy.

The proximal motive for NK action is NKG2D (AKA KLRK1), which can be triggered by viruses that increase MICA expression. NK cell depletion or NKG2D blockade, but not the depletion of T cells, stop virus clearance (Figure 4B shown). Fang makes a good case for poxvirus clearance triggered by NKG2D/KLRK1 on NK cells.

Fang M, Lanier LL, Sigal LJ. A role for NKG2D in NK cell-mediated resistance to poxvirus disease. PLoS Pathog. 2008 Feb 8;4(2):e30

Genome Modules Track Disease

The idea is alluring: take a little blood, measure some gene transcripts, and diagnose disease. The problems have been (1) identifying the genes that signal disease, and (2) overcoming natural and laboratory variations. Chaussabel and colleagues approached these problems with reliable microarray measurements of samples from 239 people and by identifying small sets, “modules”, of genes that are coordinately-expressed across a “wide range” of conditions. These modules are likely to be more reproducible than measurements of individual genes. The (seemingly arbitrary) conditions included juvenile idiopathic arthritis (47 patients), lupus (40), and type I diabetes (20), melanoma (39), immune-suppression after liver transplantation (37), and infections with E. coli (22), Staph aureus (18), and influenza (16). Genes from all 239 samples were clustered using a “K-means algorithm” with K=30 (which yields up to 30 groups) without regard to the magnitude of change in expression. The first round grouped all 8 conditions, the second round, 7, and the third, 6 (procedure). A total of nearly 5,000 transcripts in 28 modules were identified using the sample data. When the data were randomized, no modules were identified in 200 trial clusterings, suggesting that the modules reflect states of health and are not statistical artifacts. Modules range from 22 to 325 transcripts. Genes with known relationships, e.g. particular cell types or pathways, constitute about half the modules, underscoring the functional coherence.

Two examples of how health conditions change these 28 modules are shown: healthy vs. melanoma (top) and healthy vs. lupus (bottom) (from fig. 1B, red=overexpressed, blue=underexpressed). All 8 conditions are clearly distinct from healthy and distinguished from each other. The authors also identified 'biomarker' modules, e.g. M1.2 & M1.8 in melanoma or M1.7 & M3.1 in lupus in the examples shown, and made circular ('spider') graphs that can display several patients or one patient over a course of treatment.

In contrast to typical repository-biomarker analyses, very few patient samples were used to generate these modules. It will be crucial to see how they accommodate more, different samples. Also, I'm curious whether rational groupings of 'conditions', e.g., cancers or infections or autoimmune diseases, might further improve module definition. Finally, in addition to improving patient care, this information should provide invaluable insights into disease origin and progression.
Chaussabel et al. A modular analysis framework for blood genomics studies: application to systemic lupus erythematosus. Immunity. 2008 Jul;29(1):150-64.

Tolerating Aire - Out of the Thymus

The immune system protects us against infections but it must also tolerate the many different proteins that constitute our bodies. T lymphocytes of the immune system that react against the body’s own proteins could trigger autoimmunity, so they are usually deleted during their maturation in the thymus. A protein called Aire (Autoimmune regulator) turns on expression in the thymus of nearly 2,000 genes that are otherwise expressed in only a few organs and tissues, thereby exposing new T cells to these proteins and avoiding autoimmune disease. Indeed, mutations in Aire result in a broad autoimmunity against many organs and tissues. Previously, Aire was thought by many to be expressed only in the thymus.

Gardner and colleagues detected Aire in rare stromal (non-lymphoid) cells they called extrathymic Aire-expressing cells (eTACs) within the spleen, lymph nodes, and Peyer’s patches (nodes associated with the gut). These cells are located between the T and B cell areas of these organs but are highly mobile. Surprisingly, Aire turned on different genes in eTACs than were turned on in the thymus, suggesting that Aire expression in eTACs is not a ‘safety net’ for T cells that accidentally escaped the thymus but rather acts to delete additional autoreactive T cells. In eTACS, Aire turned on fewer than 200 genes, but several of these are suspected to be involved in autoimmunity, including desmoglein 1a (in pemphigous foliaceus) and the N-methyl-D-aspartate receptor 2C (in lupus). In an animal model of type 1 diabetes, eTACs expressing glucose-6-phosphate-related protein deleted T cells specific for this protein and prevented onset of this autoimmune disease (Fig. 2f shown, open squares represent mice expressing glucose-6-phosphatase-related Adig in eTACs).

These findings suggest that manipulating eTACs is a promising approach to therapy for autoimmune disease. Comparing eTACs in healthy people and autoimmune patients might also help identify deficiencies that can be corrected to prevent or ameliorate disease.

Gardner JM, Devoss JJ, Friedman RS, Wong DJ, Tan YX, Zhou X, Johannes KP, Su MA, Chang HY, Krummel MF, Anderson MS. Deletional tolerance mediated by extrathymic Aire-expressing cells. Science. 2008 Aug 8;321(5890):843-7.

Homozygosity mapping: Autism in the family

Most genes identified in genome-wide analyses contribute only modestly to disease risk or protection, with already low relative risks changed only ~20%, and taken together probably contribute to a minority of disease cases. Morrow and colleagues took a different approach, believing that analyzing individual families might reveal higher-risk genes for autism spectrum disorders (ASDs). They noted that offspring of first-cousins have twice as many neurological birth defects, including ASDs.

They also noted that a significant involvement of autosomal recessive genes would be 'signaled' by change in the male-to-female ratio (because the M:F ratio is typically 1:1 for autosomal recessive traits vs. male-dominant ASD). Indeed, among the 104 families recruited for the study, the M:F ratio was 2.6 for the offspring of the 88 consanguineous families (cousins) vs. 7.4 for the non-consanguineous.

They genotyped using arrays of SNPs and BACs, the latter validated for comparative genomic hybridization (CGH) detection of copy number variation (CNV) due to deletions, etc. Few de novo (not inherited) CNVs were detected. Large deletions on both DNA strands of the affected child (homozygous), and one strand of each parent (hemizygous), were found in 5 of the 78 consanguineous families but not among any of the other ~400 ASD cases or ~2,400 controls. In the figure (from Figure 1C), the SNPs spanning a large deletion in the AU-3100 pedigree is shown: the parents 3103 & 3104 (top and second) are hemizygous as is one child (3102, third down) but the other child, 3101 (bottom), who is homozygous for the deleted chromosome, has autism and seizures.

The investigators were surprised to find 3 genes linked to the 2 largest deletions were previously identified as being regulated by neural activity, and thus candidates for involvement with learning. Mutations in one of these genes, NHE9, were detected in non-consanguineous families and associated with additional neurological disorders (epilepsy) and learning delays (language acquisition).

EM Morrow et al. Identifying autism loci and genes by tracing recent shared ancestry. Science. 2008 Jul 11;321(5886):218-23.