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Over the past decade there has been increasing interest in the involvement of the microbiota-gut-brain axis in mental health. However, there are major gaps in our knowledge regarding the complex signaling systems through which gut microbes modulate the CNS. The immune system is a recognized mediator in the bidirectional communication continuously occurring between gut and brain. We previously demonstrated that Lactobacillus rhamnosus JB-1 (JB-1), a bacterial strain that has anxiolytic- and antidepressant-like effects in mice, modulates the immune system through induction of immunosuppressive T regulatory cells. Here we examined a potential causal relationship between JB-1 induced regulatory T cells and the observed effects on behaviour. We found that depletion of regulatory T cells, via treatment with monoclonal antibody against CD25, inhibited the antidepressant- and anxiolytic-like effects induced by 4-week oral administration of JB-1 in mice. Ly6Chi monocytes were found to be decreased in JB-1 fed mice with intact regulatory T cells, but not in JB-1 fed mice following depletion. Furthermore, adoptive transfer of CD4+CD25+ cells, from JB-1 treated donor mice, but not from controls, induced antidepressant- and anxiolytic-like effects in recipient mice. Ly6Chi monocytes were also significantly decreased in mice receiving CD4+CD25+ cells from JB1 fed donors. This study identifies cells within the CD4+CD25+ population, most likely regulatory T cells, as both necessary and sufficient in JB-1-induced antidepressant- and anxiolytic-like effects in mice, providing novel mechanistic insight into microbiota-gut-brain communication in addition to highlighting the potential for immunotherapy in psychiatric disorders.

In mammals, early-life probiotic supplementation is a promising tool for preventing unfavourable, gut microbiome-related behavioural, immunological, and aromatic amino acid alterations later in life. In laying hens, feather-pecking behaviour is proposed to be a consequence of gut-brain axis dysregulation. Lactobacillus rhamnosus decreases stress-induced severe feather pecking in adult hens, but whether its effect in pullets is more robust is unknown. Consequently, we investigated whether early-life, oral supplementation with a single Lactobacillus rhamnosus strain can prevent stress-induced feather-pecking behaviour in chickens. To this end, we monitored both the short- and long-term effects of the probiotic supplement on behaviour and related physiological parameters. We hypothesized that L. rhamnosus would reduce pecking behaviour by modulating the biological pathways associated with this detrimental behaviour, namely aromatic amino acid turnover linked to neurotransmitter production and stress-related immune responses. We report that stress decreased the proportion of cytotoxic T cells in the tonsils (P = 0.047). Counteracting this T cell depression, birds receiving the L. rhamnosus supplementation significantly increased all T lymphocyte subset proportions (P L. rhamnosus supplementation on the immune system and the turnover of the serotonin precursor tryptophan. Our findings indicate that L. rhamnosus exerts a transient, beneficial effect on the immune response and tryptophan catabolism in pullets.

Intestinal bacteria have diverse and complex influence on their host. Evidence is accumulating that this may be mediated in part by bacterial extracellular membrane vesicles (MV), nanometer-sized particles important for intercellular communication. Little is known about the composition of MV from gram-positive beneficial bacteria nor how they interact with intestinal epithelial cells (IEC). Here we demonstrate that MV from Lacticaseibacillus rhamnosus JB-1 are endocytosed in a likely clathrin-dependent manner by both mouse and human IEC in vitro and by mouse IEC in vivo. We further show that JB-1 MV contain lipoteichoic acid (LTA) which activates Toll-like receptor 2 (TLR2) and induces immunoregulatory interleukin-10 expression by dendritic cells in an internalization-dependent manner. By contrast, neither LTA nor TLR2 appear to be required for JB-1 MV endocytosis by IEC. These results demonstrate a novel mechanism by which bacterial MV can influence host physiology and suggest one potential route for beneficial influence of certain bacteria and probiotics.

Additional file 1: Supplemental Data. Representative photos of femurs from 6-month old 3xTg-AD mice and WT controls treated with cyclophosphamide or vehicle.

Gut microbiota have myriad roles in host physiology, development, and immunity. Though confined to the intestinal lumen by the epithelia, microbes influence distal systems via poorly characterized mechanisms. Recent work has considered the role of extracellular vesicles in interspecies communication, but whether they are involved in systemic microbe-host interaction is unclear. Here, we show that distinctive nanoparticles can be isolated from mouse blood within 2.5 h of consuming Lacticaseibacillus rhamnosus JB-1. In contrast to blood nanoparticles from saline-fed mice, they reproduced lipoteichoic acid-mediated immune functions of the original bacteria, including activation of TLR2 and increased IL-10 expression by dendritic cells. Like the fed bacteria, they also reduced IL-8 induced by TNF in an intestinal epithelial cell line. Though enriched for host neuronal proteins, these isolated nanoparticles also contained proteins and viral (phage) DNA of fed bacterial origin. Our data strongly suggest that oral consumption of live bacteria rapidly leads to circulation of their membrane vesicles and phages and demonstrate a nanoparticulate pathway whereby beneficial bacteria and probiotics may systemically affect their hosts.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background Circulating autoantibodies and sex-dependent discrepancy in prevalence are unexplained phenomena of Alzheimer’s disease (AD). Using the 3xTg-AD mouse model, we reported that adult males show early manifestations of systemic autoimmunity, increased emotional reactivity, enhanced expression of the histone variant macroH2A1 in the cerebral cortex, and loss of plaque/tangle pathology. Conversely, adult females display less severe autoimmunity and retain their AD-like phenotype. This study examines the link between immunity and other traits of the current 3xTg-AD model. Methods Young 3xTg-AD and wild-type mice drank a sucrose-laced 0.4 mg/ml solution of the immunosuppressant cyclophosphamide on weekends for 5 months. After behavioral phenotyping at 2 and 6 months of age, we assessed organ mass, serologic markers of autoimmunity, molecular markers of early AD pathology, and expression of genes associated with neurodegeneration. Results Chronic immunosuppression prevented hematocrit drop and reduced soluble Aβ in 3xTg-AD males while normalizing the expression of histone variant macroH2A1 in 3xTg-AD females. This treatment also reduced hepatosplenomegaly, lowered autoantibody levels, and increased the effector T cell population while decreasing the proportion of regulatory T cells in both sexes. Exposure to cyclophosphamide, however, neither prevented reduced brain mass and BDNF expression nor normalized increased tau and anxiety-related behaviors. Conclusion The results suggest that systemic autoimmunity increases soluble Aβ production and affects transcriptional regulation of macroH2A1 in a sex-related manner. Despite the complexity of multisystem interactions, 3xTg-AD mice can be a useful in vivo model for exploring the regulatory role of autoimmunity in the etiology of AD-like neurodegenerative disorders.

Chronic social defeat (CSD) in mice has been suggested as a model for studying post-traumatic stress disorder (PTSD). Our previous work indicated that exposure to Lactobacillus rhamnosus JB-1 (JB-1) during CSD can attenuate subsequent behavioural and immune disruption, suggesting a potential for microbe based therapeutic approaches in PTSD. In the current study, we assessed the ability of JB-1 to mitigate the behavioral consequences of CSD when treatment is instigated in the early post-stress period and compared the probiotic effects with those of the selective serotonin reuptake inhibitor (SSRI), sertraline. JB-1 or sertraline were administered orally 48 h following 10-days of CSD in male C57BL/6 mice. Contrary to our hypothesis of a beneficial effect, 30 days of treatment with either JB-1 or sertraline increased the persistence of both aggressor avoidance and reduced sociability in defeated mice. This was accompanied by lower hippocampal mRNA expression for genes related to fear memory. Defeated mice treated with either JB-1 or sertraline also exhibited systemic immune changes, with a decrease in Th1 cells, activated monocytes, and the monocyte chemoattractant CCL2. This study identifies potentially detrimental effects of both JB-1 and sertraline if administered in the early post-trauma period and suggests caution should be applied when considering psychobiotic or SSRI based approaches for early intervention in trauma related psychiatric disorders.

There is increasing awareness of the need to consider potential long-term effects of antibiotics on the health of children. In addition to being associated with immune and metabolic diseases, there is evidence that early-life antibiotic exposure can affect neurodevelopment. Here we investigated the effect of low dose of penicillin V on mice when administered for 1 week immediately prior to weaning. We demonstrated that exposure to the antibiotic during the pre-weaning period led to long-term changes in social behaviour, but not anxiety-like traits, in male mice only. The change in behaviour of males was associated with decreased hippocampal expression of AVPR1A and AVPR1B while expression of both receptors was increased in females. Spleens of male mice also showed an increase in the proportion of activated dendritic cells and a corresponding decrease in regulatory T cells with penicillin exposure. All changes in brain, behaviour and immune cell populations, associated with penicillin exposure, were absent in mice that received L. rhamnosus JB-1 supplementation concurrent with the antibiotic. Our study indicates that post-natal exposure to a clinically relevant dose of antibiotic has long-term, sex dependent effects on the CNS and may have implications for the development of neuropsychiatric disorders. Importantly, we also provide further evidence that probiotic based strategies may be of use in counteracting detrimental effects of early-life antibiotics on neurodevelopment.

There has been increased interest in the use of dietary ingredients, including prebiotics such as human‐milk oligosaccharides (HMOs), as therapeutic strategies for food allergy. Understanding the mechanisms underlying the beneficial effects of HMOs is important to realizing their therapeutic potential. Here we demonstrate that the HMO, 6’‐sialyllactose (6’SL) inhibited chemokine (IL‐8 and CCL20) release from T‐84 and HT‐29 cells stimulated with antigen–antibody complex, TNFα or PGE2; an effect that was PPARγ dependent and associated with decreased activity of the transcription factors AP‐1 and NFκB. In contrast, 2’‐fucosyllactose (2’FL) selectively inhibited CCL20 release in response to antigen antibody complex in a PPARγ independent manner. This study reinforces the concept that structurally different oligosaccharides have distinct biological activities and identifies, for the first time, that the HMOs, 6’SL, and 2’FL, modulate human epithelial cell responses related to allergic disease. These findings encourage further investigation of the therapeutic potential of specific HMOs in food allergy. This study provides evidence for direct effects of HMOs in addition to their prebiotic role and demonstrates, for the first time, modulation of Ag–IgE complex activation of human epithelial cells that may have important implications for food‐allergy. The study also reinforces the concept that structurally different oligosaccharides have distinct biological activities. In determining the composition of infant formula, addition of oligosaccharides with specific structures may provide direct modulation of immune responses and potentially attenuate symptoms or development of food allergy.

Over the past decade there has been increasing interest in the involvement of the microbiota-gut-brain axis in mental health. However, there are major gaps in our knowledge regarding the complex signaling systems through which gut microbes modulate the CNS. The immune system is a recognized mediator in the bidirectional communication continuously occurring between gut and brain. We previously demonstrated that Lactobacillus rhamnosus JB-1 (JB-1), a bacterial strain that has anxiolytic- and antidepressant-like effects in mice, modulates the immune system through induction of immunosuppressive T regulatory cells. Here we examined a potential causal relationship between JB-1 induced regulatory T cells and the observed effects on behaviour. We found that depletion of regulatory T cells, via treatment with monoclonal antibody against CD25, inhibited the antidepressant- and anxiolytic-like effects induced by 4-week oral administration of JB-1 in mice. Ly6C

Growing evidence suggests that environmental disruptors of maternal microbes may have significant detrimental consequences for the developing fetus. Antibiotic exposure during early life can have long-term effects on neurodevelopment in mice and humans. Here we explore whether exposure to low-dose penicillin during only the last week of gestation in mice has long-term effects on offspring behaviour, brain, immune function, and gut microbiota. We found that this treatment had sex-specific effects in the adult mouse offspring. Female, but not male, mice demonstrated decreased anxiety-like behaviours, while male, but not female, mice had abnormal social behaviours which correlated with altered brain expression of AVPR1A, AVPR1B, and OXTR, and decreases in the balance of splenic FOXP3+ regulatory T cells. Prenatal penicillin exposure also led to distinct microbiota compositions that clustered differently by sex. These data suggest that exposure of pregnant mice to even a low dose of penicillin through only the last week before birth is nonetheless sufficient to induce long-term sex-specific developmental changes in both male and female offspring.

There has been increased interest in the use of dietary ingredients, including prebiotics such as human-milk oligosaccharides (HMOs), as therapeutic strategies for food allergy. Understanding the mechanisms underlying the beneficial effects of HMOs is important to realizing their therapeutic potential. Here we demonstrate that the HMO, 6'-sialyllactose (6'SL) inhibited chemokine (IL-8 and CCL20) release from T-84 and HT-29 cells stimulated with antigen-antibody complex, TNFα or PGEThis study provides evidence for direct effects of HMOs in addition to their prebiotic role and demonstrates, for the first time, modulation of Ag-IgE complex activation of human epithelial cells that may have important implications for food-allergy. The study also reinforces the concept that structurally different oligosaccharides have distinct biological activities. In determining the composition of infant formula, addition of oligosaccharides with specific structures may provide direct modulation of immune responses and potentially attenuate symptoms or development of food allergy.

Background Stress-related disorders involve systemic alterations, including disruption of the intestinal microbial community. Given the putative connections between the microbiota, immunity, neural function, and behaviour, we investigated the potential for microbe-induced gut-to-brain signalling to modulate the impact of stress on host behaviour and immunoregulation. Methods Male C57BL/6 mice treated orally over 28 days with either Lactobacillus rhamnosus (JB-1) ™ or vehicle were subjected to chronic social defeat and assessed for alterations in behaviour and immune cell phenotype. 16S rRNA sequencing and mass spectrometry were employed to analyse the faecal microbial community and metabolite profile. Results Treatment with JB-1 decreased stress-induced anxiety-like behaviour and prevented deficits in social interaction with conspecifics. However, JB-1 did not alter development of aggressor avoidance following social defeat. Microbial treatment attenuated stress-related activation of dendritic cells while increasing IL-10+ regulatory T cells. Furthermore, JB-1 modulated the effect of stress on faecal metabolites with neuroactive and immunomodulatory properties. Exposure to social defeat altered faecal microbial community composition and reduced species richness and diversity, none of which was prevented by JB-1. Stress-related microbiota disruptions persisted in vehicle-treated mice for 3 weeks following stressor cessation. Conclusions These data demonstrate that despite the complexity of the gut microbiota, exposure to a single microbial strain can protect against certain stress-induced behaviours and systemic immune alterations without preventing dysbiosis. This work supports microbe-based interventions for stress-related disorders. Electronic supplementary material The online version of this article (doi:10.1186/s12916-016-0771-7) contains supplementary material, which is available to authorized users.

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.

Citation Mian MF, Ashkar AA. Induction of innate immune responses in the female genital tract: friend or foe of HIV-1 infection? Am J Reprod Immunol 2011; 65: 344–351 Heterosexual transmission of HIV-1 and HSV-2 across the genital tract epithelial tissue is one of the primary routes for dissemination of these viral infections. Mucosal innate immunity is the first line of defense against invading pathogens. A vast majority of mucosal HIV-1 exposures do not result in productive infections which may indicate that the innate mucosal immune system is highly protective. It has been shown that Toll-like receptors (TLR)-induced innate antiviral immunity in the genital mucosa lead to induction of type I and III interferon and prevention of HSV-2 infection. The innate antiviral function of type I and III interferons and other innate factors at genital mucosa against HIV-1 is not well defined. In this review, we summarize our current understanding and advances of the innate mucosal response to genital viral infections, including HIV-1 and HSV-2, focusing on those factors that may prevent or accelerate initial infection. Understanding how each of these components contributes to mucosal innate antiviral immunity may lead to the development of novel and effective strategies to use microbicides or antiviral agents to control HIV-1 acquisition and/or transmission.

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(-/-)/γ(c)(-/-) 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.

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.

The cellular response to dsRNA or its synthetic analog polyinosinic–polycytidylic acid (poly I:C) results in IRF-3-, IRF-7- and NF-kB-mediated activation of type 1 IFNs and pro-inflammatory cytokines critical for innate antiviral immune responses. To investigate whether cigarette smoke compromises type 1 IFN signaling in humans, peripheral blood mononuclear cells (PBMCs) from non-smoking individuals were treated with smoke-conditioned media (SCM) and stimulated with poly I:C. We observed a marked attenuation of IRF-3 and NF-kB activation in PBMCs exposed to SCM compared to control PBMCs. Similarly, PBMCs from smokers or splenocytes from smoke-exposed mice also displayed marked reduction of poly I:C-induced antiviral responses compared with either non-smokers or sham-exposed mice. Cigarette smoke was found to block the production of type I IFNs following poly I:C treatment and inhibit subsequent STAT1 activation. Finally, we confirmed that inhibition of IFN-β, but not IFN-α, predominantly contributes to the cigarette smoke-mediated suppression of innate antiviral responses. These findings provide novel mechanistic insights to the susceptibility of cigarette smokers to viral infections.

Ingestion of a commensal bacteria, Lactobacillus rhamnosus JB-1, has potent immunoregulatory effects, and changes nerve-dependent colon migrating motor complexes (MMCs), enteric nerve function, and behavior. How these alterations occur is unknown. JB-1 microvesicles (MVs) are enriched for heat shock protein components such as chaperonin 60 heat-shock protein isolated from Escherichia coli (GroEL) and reproduce regulatory and neuronal effects in vitro and in vivo. Ingested labeled MVs were detected in murine Peyer's patch (PP) dendritic cells (DCs) within 18 h. After 3 d, PP and mesenteric lymph node DCs assumed a regulatory phenotype and increased functional regulatory CD4(+)25(+)Foxp3+ T cells. JB-1, MVs, and GroEL similarly induced phenotypic change in cocultured DCs via multiple pathways including C-type lectin receptors specific intercellular adhesion molecule-3 grabbing non-integrin-related 1 and Dectin-1, as well as TLR-2 and -9. JB-1 and MVs also decreased the amplitude of neuronally dependent MMCs in an ex vivo model of peristalsis. Gut epithelial, but not direct neuronal application of, MVs, replicated functional effects of JB-1 on in situ patch-clamped enteric neurons. GroEL and anti-TLR-2 were without effect in this system, suggesting the importance of epithelium neuron signaling and discrimination between pathways for bacteria-neuron and -immune communication. Together these results offer a mechanistic explanation of how Gram-positive commensals and probiotics may influence the host's immune and nervous systems.

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.

Poly I:C, a synthetic dsRNA analogue, has been used extensively for decades to study innate responses in vivo and in different cell types. We have found substantial variability while using poly I:C from different sources. In this study we found that poly I:C from 2 commercial sources induced sharply opposite responses in myeloid and fibroblasts, depending on the length of the poly I:C. Although short poly I:C (∼1–1.5 kb) induced greater amounts of TNF-α, IL-8, and IFN-β and a stronger antiviral response in myeloid cells, it was a poor inducer in fibroblasts. By contrast, long poly I:C (>5 kb) preferentially elicited higher cytokine and antiviral responses in fibroblasts and showed diminished responses in myeloid cells. Poly I:C activated NF-κB and STAT-1 signaling in a length- and cell-type–dependent fashion. Mechanistically, short poly I:C was better internalized in the myeloid cells and long poly I:C in the fibroblasts. Finally, long poly I:C required SR-A, whereas short poly I:C required RIG-I and Raftlin. We provide evidence that the length of dsRNA drives distinct innate responses in different cell lineages. These findings may augment in selecting the appropriate poly I:C type to design cell-type–specific potent adjuvants for vaccines against infectious diseases or cancers.

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.

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 a, interleukin 6, interleukin 8, and monocyte chemoattractant protein-1 was elevated in GECs exposed to copathogens. Inhibition of nuclear factorj Ba nd 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.

Salmonella enterica serovar Typhi (S. typhi), is a human restricted pathogen and the causal agent of typhoid fever. Although the use of antimicrobial drugs or vaccines has served as an effective therapeutics strategy against typhoid fever, the recent surge in multidrug resistant strains of S. typhi presents a major health concern worldwide. Progress on typhoid research has been limited in the past due to the lack of a suitable animal model that recapitulates the hallmark immunological features of human typhoid fever. We have recently developed a humanized immune system (HIS) mouse model that after intravenous challenge with S. typhi displayed classical manifestations of human typhoid fever including meningitis, liver pathology and mortality. Concurrent to our studies, two other groups also have developed humanized mouse models of S. typhi infections employing different protocols. All these recently adopted animal models of S. typhi infections provide promise for a new era of S. typhi research that may expedite detailed understanding of the cellular and molecular mechanisms of this bacterial infection and investigations of new antimicrobials and vaccines for effective control.

The full-length coding sequence of chicken interferon-γ (ChIFN-γ) was cloned into a baculovirus nonfusion vector, pFastBacDual, and expressed in Sf21 insect cells. Recombinant ChIFN-γ (rChIFN-γ) protein was found to be expressed both intracellularly as well as in the culture supernatants. The affinity-purified rChIFN-γ contained 14, 17, and 28 kDa proteins, possibly representing both glycosylated and nonglycosylated protein forms of ChIFN-γ. The bioactivity of rChIFN-γ was confirmed in vitro by production of nitric oxide in a chicken macrophage cell line (HD11) and antiviral activity against vesicular stomatitis virus in primary chicken embryonic fibroblast cells. Further, HD11 cells stimulated with rChIFN-γ showed significant upregulation of inducible nitric oxide synthases, IFN-γ, interleukin-1β, interleukin-12p35, signal transducers and activators of transcription 1, class II, major histocompatibility complex, transactivator, and major histocompatibility complex II-β chain (BL-B) transcripts. In conclusion, the present study provides information on the ability of functionally active rChIFN-γ expressed in a baculovirus system in inducing significant transcriptional upregulation of various immune system-related genes, including those that encode cytokines, antigen-presenting molecules, and transcription factors involved in the major histocompatibility complex and IFN-signaling pathway.

Heterosexual transmission of HIV-1 and HSV-2 across the genital tract epithelial tissue is one of the primary routes for dissemination of these viral infections. Mucosal innate immunity is the first line of defense against invading pathogens. A vast majority of mucosal HIV-1 exposures do not result in productive infections which may indicate that the innate mucosal immune system is highly protective. It has been shown that Toll-like receptors (TLR)-induced innate antiviral immunity in the genital mucosa lead to induction of type I and III interferon and prevention of HSV-2 infection. The innate antiviral function of type I and III interferons and other innate factors at genital mucosa against HIV-1 is not well defined. In this review, we summarize our current understanding and advances of the innate mucosal response to genital viral infections, including HIV-1 and HSV-2, focusing on those factors that may prevent or accelerate initial infection. Understanding how each of these components contributes to mucosal innate antiviral immunity may lead to the development of novel and effective strategies to use microbicides or antiviral agents to control HIV-1 acquisition and/or transmission.

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.

Both NK cells and IL-15 play crucial roles in innate immunity against viral infections and cancer. Cigarette smoke is known to increase susceptibility to infections and certain cancers. Interleukin (IL)-15 plays an important role in immune responses by regulating proliferation, survival and functions of NK cells. Here, we examined the impact of cigarette smoke on IL-15 production and IL-15 mediated NK cell functions in human PBMCs. We report that cigarette smoke significantly suppresses the induction of IL-15 by poly I:C in human PBMCs. Serum IL-15 levels among smokers was significantly lower than non-smokers. In contrast to a profound increases in intracellular IL-15/IL-15Ralpha in poly I:C-treated PBMCs, exposure of PBMCs to smoke-conditioned media (SCM) diminished the IL-15/IL-15Ralpha production. We examined if inhibition of IL-15 production could lead to less NK cell activation. Interestingly, SCM-treated PBMCs had diminished up-regulation of NK cell activation marker, CD69, but not NKG2D compared with controls after poly I:C stimulation. We then confirmed by using IL-15 neutralizing antibody as well as exogenous IL-15 that the ploy I:C-induced NK cells activation was IL-15 mediated. More importantly, cigarette smoke significantly impaired NK cell cytolytic potential to kill K562 cancer cells which was found to be IL-15 mediated. The inhibition of IL-15 and its regulatory NK cell activities were linked to attenuated STAT3 and STAT5, but not ERK1/2 phosphorylations. We demonstrate, for the first time, that cigarette smoke compromises IL-15 production and as a result NK cell function which could link to the higher incidence of cancers or viral infections observed among smokers.

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.

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.

Introduction: Airway smooth muscle cells (ASMC) are involved in lung inflammatory responses through alterations in secretion of chemokines and growth factors as well as proliferation or contractile functions. Thus ASMC contribute to levels of chemokines such as MCP-1 and eotaxins, that can enhance cellular infiltration, and modulators of extracellular matrix such as VEGF. Since ASMC express TLRs, and the lung mucosa is exposed to TLR ligands through environmental/infectious agents, we assessed in vitro responses of human (H) ASMC to TLR-ligands alone or in context of gp130 cytokines that are elevated during lung inflammation. Methods: HASMC were cultured and stimulated with ligands for TLR-3 (poly IC) and TLR-4 (LPS) alone and in combination with gp130 cytokine family members including Oncostatin M. Supernatants were saved for ELISA analysis and cell extracts were prepared for RNA analysis and cell signaling protein analysis by immunoblots. Results: TLR-3 ligand induced MCP-1, IP-10, IL-6 and eotaxin-3 responses by HASMC in dose dependent fashion, however reduced VEGF levels (protein and mRNA). TLR-4 ligand showed lower activity. OSM induced IL-6, MCP-1 and VEGF expression in vitro, whereas other members of the gp130 cytokine family (LIF, IL-31, IL-6) did not. Combination experiments showed that HASMC treated with 0.1 ng/ml OSM responded to poly IC or LPS at 10-fold less TLR-ligand concentration indicating enhanced sensitivity to TLR-3 and −4 ligands. Immunoblots showed that OSM stimulation in combination with either poly IC or LPS activated the NFkappa B pathway (IkB alpha degradation) to a greater extent than OSM or TLR ligand stimulus alone. Furthermore, OSM induced an increase in TLR-3 mRNA and CD14 mRNA. Conclusion: OSM increases the responses of HASMC to TLR-3 and TLR4 ligands, including NFkappa B activation, elevation of receptor chains for TLRs, and chemokine release, and thus is potentially integral to exacerbation of lung inflammatory diseases. This work was supported by the Canadian Institutes for Health Research .