Erol Fikrig

Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis.

West Nile virus (WNV), a mosquito-borne single-stranded (ss)RNA flavivirus, causes human disease of variable severity. We investigated the involvement of Toll-like receptor (Tlr) 3, which recognizes viral double-stranded (ds)RNA, on WNV infection. Tlr3-deficient (Tlr3−/−) mice were more resistant to lethal WNV infection and had impaired cytokine production and enhanced viral load in the periphery, whereas in the brain, viral load, inflammatory responses and neuropathology were reduced compared to wild-type mice. Peripheral WNV infection led to a breakdown of the blood-brain barrier and enhanced brain infection in wild-type but not in Tlr3−/− mice, although both groups were equally susceptible upon intracerebroventricular administration of the virus. Tumor necrosis factor-α receptor 1 signaling is vital for blood-brain barrier compromise upon Tlr3 stimulation by dsRNA or WNV. Collectively, WNV infection leads to a Tlr3-dependent inflammatory response, which is involved in brain penetration of the virus and neuronal injury.

The IFITM Proteins Mediate Cellular Resistance to Influenza A H1N1 Virus, West Nile Virus, and Dengue Virus

Influenza viruses exploit host cell machinery to replicate, resulting in epidemics of respiratory illness. In turn, the host expresses antiviral restriction factors to defend against infection. To find host cell modifiers of influenza A H1N1 viral infection, we used a functional genomic screen and identified over 120 influenza A virus-dependency factors with roles in endosomal acidification, vesicular trafficking, mitochondrial metabolism, and RNA splicing. We discovered that the interferon-inducible transmembrane proteins IFITM1, 2, and 3 restrict an early step in influenza A viral replication. The IFITM proteins confer basal resistance to influenza A virus but are also inducible by interferons type I and II and are critical for interferons virustatic actions. Further characterization revealed that the IFITM proteins inhibit the early replication of flaviviruses, including dengue virus and West Nile virus. Collectively this work identifies a family of antiviral restriction factors that mediate cellular innate immunity to at least three major human pathogens.

Vaccination against Lyme Disease with Recombinant Borrelia burgdorferi Outer-Surface Lipoprotein A with Adjuvant

BACKGROUND The risk of acquiring Lyme disease is high in areas in which the disease is endemic, and the development of a safe and effective vaccine is therefore important. METHODS We conducted a multicenter, double-blind, randomized trial involving 10,936 subjects who lived in areas of the United States in which Lyme disease is endemic. Participants received an injection of either recombinant Borrelia burgdorferi outer-surface lipoprotein A (OspA) with adjuvant or placebo at enrollment and 1 and 12 months later. In cases of suspected Lyme disease, culture of skin lesions, polymerase-chain-reaction testing, or serologic testing was done. Serologic testing was performed 12 and 20 months after study entry to detect asymptomatic infections. RESULTS In the first year, after two injections, 22 subjects in the vaccine group and 43 in the placebo group contracted definite Lyme disease (P=0.009); vaccine efficacy was 49 percent (95 percent confidence interval, 15 to 69 percent). In the second year, after the third injection, 16 vaccine recipients and 66 placebo recipients contracted definite Lyme disease (P<0.001); vaccine efficacy was 76 percent (95 percent confidence interval, 58 to 86 percent). The efficacy of the vaccine in preventing asymptomatic infection was 83 percent in the first year and 100 percent in the second year. Injection of the vaccine was associated with mild-to-moderate local or systemic reactions lasting a median of three days. CONCLUSIONS Three injections of vaccine prevented most definite cases of Lyme disease or asymptomatic B. burgdorferi infection.

RNA interference screen for human genes associated with West Nile virus infection.

West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus–host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV–human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.

Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice.

The Lyme disease vaccine is based on the outer-surface lipoprotein (OspA) of the pathogen Borrelia burgdorferi, and 95% of vaccine recipients develop substantial titers of antibodies against OspA. Here, we identified seven individuals with very low antibody titers after vaccination (low responders). The macrophages of low responders produced less tumor necrosis factor-α and interleukin-6 after OspA stimulation and had lower cell-surface expression of Toll-like receptor (TLR) 1 as compared to normal cells, but normal expression of TLR2. TLRs activate innate responses to pathogens, and TLR2 recognizes lipoproteins and peptidoglycan (PGN). After OspA immunization, mice genetically deficient in either TLR2 (TLR2−/−) or TLR1 (TLR1−/−) produced low titers of antibodies against OspA. Notably, macrophages from TLR2−/− mice were unresponsive to OspA and PGN, whereas those from TLR1−/− mice responded normally to PGN but not to OspA. These data indicate that TLR1 and TLR2 are required for lipoprotein recognition and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.

Protection of mice against the Lyme disease agent by immunizing with recombinant OspA

Lyme borreliosis is a tick-borne illness caused by Borrelia burgdorferi. The gene for outer surface protein A (OspA) from B. burgdorferi strain N40 was cloned into an expression vector and expressed in Escherichia coli. C3H/HeJ mice actively immunized with live transformed E. coli or purified recombinant OspA protein produced antibodies to OspA and were protected from challenge with several strains of B. burgdorferi. Recombinant OspA is a candidate for a vaccine for Lyme borreliosis.

The Lyme disease agent exploits a tick protein to infect the mammalian host.

The Lyme disease agent, Borrelia burgdorferi, is maintained in a tick–mouse cycle. Here we show that B. burgdorferi usurps a tick salivary protein, Salp15 (ref. 3), to facilitate the infection of mice. The level of salp15 expression was selectively enhanced by the presence of B. burgdorferi in Ixodes scapularis, first indicating that spirochaetes might use Salp15 during transmission. Salp15 was then shown to adhere to the spirochaete, both in vitro and in vivo, and specifically interacted with B. burgdorferi outer surface protein C. The binding of Salp15 protected B. burgdorferi from antibody-mediated killing in vitro and provided spirochaetes with a marked advantage when they were inoculated into naive mice or animals previously infected with B. burgdorferi. Moreover, RNA interference-mediated repression of salp15 in I. scapularis drastically reduced the capacity of tick-borne spirochaetes to infect mice. These results show the capacity of a pathogen to use a secreted arthropod protein to help it colonize the mammalian host.

STAT3 deletion during hematopoiesis causes Crohn's disease-like pathogenesis and lethality : a critical role of STAT3 in innate immunity

Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional mediator for many cytokines and is essential for normal embryonic development. We have generated a unique strain of mice with tissue-specific disruption of STAT3 in bone marrow cells during hematopoiesis. This specific STAT3 deletion causes death of these mice within 4–6 weeks after birth with Crohns disease-like pathogenesis in both the small and large intestine, including segmental inflammatory cell infiltration, ulceration, bowel wall thickening, and granuloma formation. Deletion of STAT3 causes significantly increased cell autonomous proliferation of cells of the myeloid lineage, both in vivo and in vitro. Most importantly, Stat3 deletion during hematopoiesis causes overly pseudoactivated innate immune responses. Although inflammatory cytokines, including tumor necrosis factor α and IFN-γ, are overly produced in these mice, the NAPDH oxidase activity, which is involved in antimicrobial and innate immune responses, is inhibited. The signaling responses to lipopolysaccharide are changed in the absence of STAT3, leading to enhanced NF-κB activation. Our results suggest a model in which STAT3 has critical roles in the development and regulation of innate immunity, and deletion of STAT3 during hematopoiesis results in abnormalities in myeloid cells and causes Crohns disease-like pathogenesis.

Age-associated decrease in TLR function in primary human dendritic cells predicts influenza vaccine response.

We evaluated TLR function in primary human dendritic cells (DCs) from 104 young (age 21–30 y) and older (≥65 y) individuals. We used multicolor flow cytometry and intracellular cytokine staining of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) and found substantial decreases in older compared with young individuals in TNF-α, IL-6, and/or IL-12 (p40) production in mDCs and in TNF-α and IFN-α production in pDCs in response to TLR1/2, TLR2/6, TLR3, TLR5, and TLR8 engagement in mDCs and TLR7 and TLR9 in pDCs. These differences were highly significant after adjustment for heterogeneity between young and older groups (e.g., gender, race, body mass index, number of comorbid medical conditions) using mixed-effect statistical modeling. Studies of surface and intracellular expression of TLR proteins and of TLR gene expression in purified mDCs and pDCs revealed potential contributions for both transcriptional and posttranscriptional mechanisms in these age-associated effects. Moreover, intracellular cytokine production in the absence of TLR ligand stimulation was elevated in cells from older compared with young individuals, suggesting a dysregulation of cytokine production that may limit further activation by TLR engagement. Our results provide evidence for immunosenescence in DCs; notably, defects in cytokine production were strongly associated with poor Ab response to influenza immunization, a functional consequence of impaired TLR function in the aging innate immune response.

Inhibition of Th1 Differentiation by IL-6 Is Mediated by SOCS1

Interleukin 6 (IL-6) is a cytokine produced by immune and nonimmune cells and exhibits functional pleiotropy and redundancy. IL-6 plays an important role in the differentiation of several cell types. Here, we describe a novel function of IL-6: the negative regulation of CD4+ Th1 cell differentiation. While IL-6-directed CD4+ Th2 differentiation is mediated by IL-4, inhibition of Th1 differentiation by IL-6 is independent of IL-4. IL-6 upregulates suppressor of cytokine signaling 1 (SOCS1) expression in activated CD4+ T cells, thereby interfering with signal transducer and activator of transcription 1 (STAT1) phosphorylation induced by interferon gamma (IFNgamma). Inhibition of IFNgamma receptor-mediated signals by IL-6 prevents autoregulation of IFNgamma gene expression by IFNgamma during CD4+ T cell activation, thereby preventing Th1 differentiation. Thus, IL-6 promotes CD4+ Th2 differentiation and inhibits Th1 differentiation by two independent molecular mechanisms.