Friday, October 30, 2009

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, 68 (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.

Wednesday, October 7, 2009

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.

Monday, September 21, 2009

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.

Monday, April 6, 2009

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.

Thursday, February 26, 2009

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