M. Firoz Mian

Cutting Edge: FimH Adhesin of Type 1 Fimbriae Is a Novel TLR4 Ligand

Several TLR ligands of bacterial origin induce innate immune responses. Although FimH, the adhesin portion of type 1 fimbria, plays an important role in the pathogenicity of some Gram-negative bacteria, its ability to stimulate the innate immune system via TLR signaling remains unclear. In this study we report that FimH induces potent innate responses in a MyD88-dependent fashion. The FimH-induced innate activity was restricted to cells expressing TLR4. In addition, FimH was able to bind directly to TLR4. More importantly, cells unresponsive to LPS were responsive to FimH and the presence or absence of MD-2 and CD14 had no effect on FimH activity. Our data suggest that TLR4 is a functional receptor for the adhesin portion of bacterial type 1 fimbria.

Impairment of human NK cell cytotoxic activity and cytokine release by cigarette smoke

NK cells play essential roles in innate host defense against microbial infections and tumor surveillance. Although evidence suggests that smoking has adverse effects on the immune system, little is known about whether smoking compromises NK cell effector functions. In this study, we show that cigarette smoke‐conditioned medium (SCM) dose‐dependently inhibits in vitro IFN‐γ production by polyinosinic:polycytidylic acid (poly I:C)‐activated PBMC and NK cells isolated from nonsmoking individuals. Similarly, SCM attenuated poly I:C‐induced TNF‐α production by PBMC and NK cells. The inhibitory effect of cigarette smoke on TNF‐α production was reversible. PBMC and NK cells isolated from smokers displayed significant reduction of IFN‐γ and TNF‐α secretions compared with nonsmokers in response to poly I:C activation. We further observed that SCM attenuated NK cell cytotoxic activity, which was associated with decreased up‐regulation of perforin expression. Attenuated cytotoxic activity was also observed in PBMCs isolated from smokers. Finally, anti‐IL‐12 mAb‐blocking data revealed that an attenuation of IFN‐γ production by PBMC was indirect, likely via attenuation of IL‐12 production, and the effect on NK cells was IL‐12‐independent. Our data indicate that cigarette smoke compromises function of human NK cells. This may contribute to a higher incidence of viral infections and cancer among smokers.

HIV-1 gp120 Induces TLR2- and TLR4-Mediated Innate Immune Activation in Human Female Genital Epithelium

Although women constitute half of all HIV-1–infected people worldwide (UNAIDS World AIDS Day Report, 2011), the earliest events in the female reproductive tract (FRT) during heterosexual HIV-1 transmission are poorly understood. Recently, we demonstrated that HIV-1 could directly impair the mucosal epithelial barrier in the FRT. This suggested that the HIV-1 envelope glycoprotein gp120 was being recognized by a membrane receptor on genital epithelial cells, leading to innate immune activation. In this study, we report that pattern-recognition receptors TLR2 and -4 bind to HIV-1 gp120 and trigger proinflammatory cytokine production via activation of NF-κB. The gp120–TLR interaction also required the presence of heparan sulfate (HS). Bead-binding assays showed that gp120 can bind to HS, TLR2, and TLR4, and studies in transfected HEK293 cells demonstrated that HS and TLR2 and -4 were necessary to mediate downstream signaling. Exposure to seminal plasma from HIV-1–infected and uninfected men with gp120 added to it induced a significant proinflammatory cytokine response from genital epithelial cells and disruption of tight junctions, indicating a role for gp120 in mucosal barrier disruption during HIV-1 heterosexual transmission. These studies provide, for the first time to our knowledge, a possible mechanism by which HIV-1 gp120 could directly initiate innate immune activation in the FRT during heterosexual transmission.

Critical Role of Natural Killer Cells in Lung Immunopathology During Influenza Infection in Mice

Influenza viral infection results in excessive pulmonary inflammation that has been linked to the damage caused by immune responses and viral replication. The multifunctional cytokine interleukin (IL-15), influences the proliferation and maintenance of immune cells such as CD8(+) T cells and natural killer (NK) cells. Here we show that IL-15(-/-) mice are protected from lethal influenza infection. Irrespective of the mouse strains, the protection observed was linked to the lack of NK cells. Increased survival in the IL-15(-/-) or NK1.1(+) cell-depleted wild-type mice was associated with significantly lower lung lesions as well as decreased mononuclear cells and neutrophils in the airway lumen. Levels of interleukin 10 were significantly higher and levels of proinflammatory cytokines, including interleukin 6 and interleukin 12, were significantly lower in the bronchoalveolar lavage fluid from IL-15(-/-) and NK1.1(+) cell-depleted wild-type mice than in that from control mice. Our data suggest that NK cells significantly augment pulmonary inflammation, contributing to the pathogenesis of influenza infection.

Structural & functional consequences of chronic psychosocial stress on the microbiome & host

INTRODUCTION Given the lasting impact of psychological distress on behavior, along with the role of the microbiome in neurobehavioral development, we sought to examine the relationship between the microbiota and stress-induced behavioral deficits. METHODS Male C57BL/6 mice exposed to chronic social defeat were subjected to behavioral analysis and profiling of the intestinal microbiome. Mice were also analyzed for phenotypic and functional immune changes. A computational approach on 16S rRNA marker gene sequences was used to predict functional changes in the metagenome as a consequence of structural shifts in the microbiota. RESULTS Chronic social defeat induced behavioral changes that were associated with reduced richness and diversity of the gut microbial community, along with distinct shifts at the level of operational taxonomic units (OTU) across phyla. The degree of deficits in social, but not exploratory behavior was correlated with group differences between the microbial community profile. In silico analysis predicted a shift in the functional profile of the microbiome: defeated mice exhibited reduced functional diversity and a lower prevalence of pathways involved in the synthesis and metabolism of neurotransmitter precursors and short-chain fatty acids. Defeated mice also exhibited sustained alterations in dendritic cell activation, and transiently elevated levels of IL-10+ T regulatory cells that were suppressed over time. CONCLUSIONS This study indicates that stress-induced disruptions in neurologic function are associated with altered immunoregulatory responses and complex OTU-level shifts in the microbiota. It is thus suggested that a dysbiotic state, along with specific changes in microbial markers, may predict the onset of adverse neurocognitive deficits commonly observed following exposure to severe stressors. The data also predict novel pathways that might underlie microbiota-mediated effects on brain and behavior, thus presenting targets for investigations into mechanisms and potential therapy.

Humanized mice are susceptible to Salmonella typhi infection

Salmonella enterica serovar Typhi is a pathogen that only infects humans. Currently, there is no animal model for studying this pathogen. Recently, alymphoid RAG-2−/−/γc−/− mice engrafted with human leukocytes, known as humanized mice, have been successfully utilized to develop experimental models for several human-specific viral infections, including HIV, human-like dengue fever and hepatitis C virus. Little is known about the usefulness and feasibility of the humanized mouse model for the study of human-specific bacterial pathogens, such as S. typhi. The aim of this study was to determine if Salmonella enterica serovar Typhi could establish productive infection in humanized mice. Here we report that intravenous inoculation of S. typhi into humanized mice, but not controls, established S. typhi infections. High bacterial loads were found in the liver, spleen, blood and bone marrow of mice reconstituted with human leukocytes, but not in the unreconstituted control mice. Importantly, S. typhi-infected humanized mice lost significant body weight, and some of the infected mice displayed neurological symptoms. Our data suggest, for the first time, that humanized mice are susceptible to S. typhi challenge and that this model can be utilized to study the pathogenesis of S. typhi to develop novel therapeutic strategies.

FimH Can Directly Activate Human and Murine Natural Killer Cells via TLR4

Although the importance of natural killer (NK) cells in innate immune responses against tumors or viral infections are well documented, their ability to directly recognize pathogens is less well defined. We have recently reported FimH, a bacterial fimbrial protein, as a novel Toll-like receptor (TLR)4 ligand that potently induces antiviral responses. Here, we investigated whether FimH either directly or indirectly can activate human and murine NK cells. We demonstrate that FimH potently activates both human and murine NK cells in vitro to induce cytokines [interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha] and cytotoxicity. Importantly, NK cells directly recognize FimH-expressing pathogens as FimH(+), but not FimH(-), bacteria were able to activate human NK cells. FimH activation of NK cells required TLR4 and MyD88 signaling, as NK cells from both TLR4(-/-) and MyD88(-/-) mice as well as human NK-92 cells, which lack TLR4, were all unresponsive to FimH. In addition, TLR4 neutralization significantly abrogated the response of human NK cells to FimH. Activation of purified NK cells by FimH was independent of lipopolysaccharide (LPS) or other bacterial contaminations. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to FimH via TLR4-MyD88 pathways to aid innate protection against cancer or microbial infections.

IL-15 Can Signal via IL-15Rα, JNK, and NF-κB To Drive RANTES Production by Myeloid Cells

IL-15 plays many important roles within the immune system. IL-15 signals in lymphocytes via trans presentation, where accessory cells such as macrophages and dendritic cells present IL-15 bound to IL-15Rα in trans to NK cells and CD8+ memory T cells expressing IL-15/IL-2Rβ and common γ chain (γc). Previously, we showed that the prophylactic delivery of IL-15 to Rag2−/−γc−/− mice (mature T, B, and NK cell negative) afforded protection against a lethal HSV-2 challenge and metastasis of B16/F10 melanoma cells. In this study, we demonstrated that in vivo delivery of an adenoviral construct optimized for the secretion of human IL-15 to Rag2−/−γc−/− mice resulted in significant increases in spleen size and cell number, leading us to hypothesize that IL-15 signals differently in myeloid immune cells compared with lymphocytes, for which IL-15/IL-2Rβ and γc expression are essential. Furthermore, treatment with IL-15 induced RANTES production by Rag2−/−γc−/− bone marrow cells, but the presence of γc did not increase bone marrow cell sensitivity to IL-15. This IL-15–mediated RANTES production by Rag2−/−γc−/− bone marrow cells occurred independently of the IL-15/IL-2Rβ and Jak/STAT pathways and instead required IL-15Rα signaling as well as activation of JNK and NF-κB. Importantly, we also showed that the trans presentation of IL-15 by IL-15Rα boosts IL-15–mediated IFN-γ production by NK cells but reduces IL-15–mediated RANTES production by Rag2−/−γc−/− myeloid bone marrow cells. Our data clearly show that IL-15 signaling in NK cells is different from that of myeloid immune cells. Additional insights into IL-15 biology may lead to novel therapies aimed at bolstering targeted immune responses against cancer and infectious disease.

FimH, a TLR4 ligand, induces innate antiviral responses in the lung leading to protection against lethal influenza infection in mice

Fimbriae H protein (FimH) is a novel TLR4 ligand that has been shown to stimulate the innate immune system and elicits protective responses against bacterial and viral infections. Here, we evaluated the protective role of local delivery of FimH against influenza A infection in a mouse model. We show that intranasal delivery of FimH prior to lethal challenge with influenza A virus, resulted in decreased morbidity and mortality in wild-type, but not TLR4(-/-), mice. Importantly, FimH was able to reduce the early viral burden in the lung leading to minimal cell infiltration into the airway lumen and reduced pulmonary pathology following infection in wild type mice compared to TLR4(-/-) mice. Local delivery of FimH to C57BL/6, not TLR4(-/-), mice in a prophylactic manner increased the IL-12 and RANTES responses as well as neutrophil recruitment into the airway lumen. These effects correlate to the course of influenza infection. The FimH-mediated antiviral response against influenza virus appears to be partially dependent on alveolar macrophages. The antiviral effects are likely mediated by the innate mediators (TNF-α, IL-12 or RANTES) and/or by activation of a feedback inhibition loop to curtail the pulmonary inflammation possibly be the potential mechanisms involved in FimH-mediated protection. FimH thus holds promise to be a possible prophylactic mean of control against influenza viral infection.

Endometrial Epithelial Cell Responses to Coinfecting Viral and Bacterial Pathogens in the Genital Tract Can Activate the HIV-1 LTR in an NFκB-and AP-1–Dependent Manner

BACKGROUND Sexually transmitted infections (STIs) are associated with increased human immunodeficiency virus type 1 (HIV-1) susceptibility and viral shedding in the genital tract, but the mechanisms underlying this association are poorly understood. METHODS Direct activation of HIV long terminal repeats (LTRs), a proxy measure for HIV-1 replication, was measured after treatment of 1G5 T cells with Toll-like receptor (TLR) ligands, herpes simplex virus type 1 or 2 (HSV-1/2), or Neisseria gonorrhoeae. For indirect activation, 1G5 T cells were incubated with supernatants from female primary genital epithelial cells (GECs) previously exposed to these agents. Proinflammatory cytokines and chemokines were measured in GEC supernatants. Proinflammatory pathways were blocked to determine the mechanisms of direct and indirect HIV-LTR activation. RESULTS HSV-1/2, N. gonorrhoeae, and TLR ligands FimH (TLR-4), flagellin (TLR-5), and Poly (I:C) (TLR-3) directly induced HIV-LTR activation in 1G5 T cells. Supernatants collected from GECs incubated with these agents indirectly induced HIV-LTR activation. Production of tumor necrosis factor α, interleukin 6, interleukin 8, and monocyte chemoattractant protein-1 was elevated in GECs exposed to copathogens. Inhibition of nuclear factor κB and activator protein-1 (AP-1) signaling pathways in 1G5 T cells abrogated both direct and indirect HIV-LTR activation. CONCLUSIONS STIs may increase HIV-1 replication in the female genital tract via proinflammatory signaling pathways directly and indirectly via their effects on GECs. This increased HIV-1 replication may enhance sexual and vertical HIV transmission.