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Intestinal T helper type 2 (Th2) immunity in food allergy results in IgG1 and IgE production, and antigen re-exposure elicits responses such as anaphylaxis and eosinophilic inflammation. Although interleukin-4 (IL-4) is critically required for allergic sensitization, the source and control of IL-4 during the initiation of Th2 immunity in vivo remains unclear. Non-intestinal and non-food allergy systems have suggested that natural killer-like T (NKT) or γδ T-cell innate lymphocytes can supply the IL-4 required to induce Th2 polarization. Group 2 innate lymphoid cells (ILCs) are a novel IL-4-competent population, but their contribution to initiating adaptive Th2 immunity is unclear. There are also reports of IL-4-independent Th2 responses. Here, we show that IL-4-dependent peanut allergic Th2 responses are completely intact in NKT-deficient, γδ T-deficient or ILC-deficient mice, including antigen-specific IgG1/IgE production, anaphylaxis, and cytokine production. Instead, IL-4 solely from CD4(+) Th cells induces full Th2 immunity. Further, CD4(+) Th cell production of IL-4 in vivo is dependent on OX40L, a costimulatory molecule on dendritic cells (DCs) required for intestinal allergic priming. However, both Th2 cells and ILCs orchestrated IL-13-dependent eosinophilic inflammation. Thus, intestinal Th2 priming is initiated by an autocrine/paracrine acting CD4(+) Th cell-intrinsic IL-4 program that is controlled by DC OX40L, and not by NKT, γδ T, or ILC cells.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Natural Killer cells can distinguish between healthy and malignant cells and have the unique ability to lyse tumour cells without prior sensitization. Differences between murine and human NK cells complicate the translation of this knowledge into useful therapeutics. Humanized mouse models that support the development of human leukocytes are a promising avenue of research that aims to address this problem. Here we provide an in-depth phenotypic analysis of human NK cells in Balb/c Rag2(-/-)γ(c)(-/-) mice reconstituted with human hematopoietic stem cells. We have examined the development of NK cells in bone marrow, thymous, spleen, lymph node (LN) and liver. Interestingly, in naive reconstituted mice, NK cells were found in thymus and LN, but not in bone marrow. These NK cells expressed several inhibitory and activating receptors needed for malignant cell detection. Furthermore, we confirm that administration of recombinant human interleukin-15 (rhIL-15) or Ad-vector expressing hIL-15 is able to significantly enhance NK cell development and maturation, particularly in bone marrow and liver, in this model. Our results suggest that human NK cells developed in mice may have phenotypes and tissue distributions similar to those seen in human., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

During activation, macrophages undergo physiological changes affecting their surface protein expression and cytokine production and have been subsequently categorized into M1 (classically-activated) and M2 (alternatively-activated) macrophages. It remains unclear which lymphocyte population provides the immune microenvironment to regulate macrophage polarization. In this study, we establish a functional and phenotypic profile of peritoneal macrophages from C57BL/6 wild-type mice. We also showed that Rag1(-/-) and Rag2(-/-)γc(-/-) mice have similar, exaggerated M1 characteristics in comparison to control mice, suggesting that NK and/or NK-T cells may not be essential in this process. By controlling for environmental factors, we determine that lymphocyte-derived cytokines, rather than inherent properties of macrophages themselves, are crucial for their regulation. Lastly, we report that macrophages from CD4(-/-) mice display an M1 profile, suggesting that CD4(+) T-cells play a dominant role over other lymphocyte populations in providing the cytokine environment for regulating macrophages towards an M2 profile under normal wild-type conditions., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Type I interferon (IFN) signalling, NK cells and NK cell-derived IFN-γ are critical in the early control of genital HSV-2 infection. We have recently reported that NK cells are the source of early IFN-γ in the genital tract in response to HSV-2. However, the response of NK cells to genital HSV-2 infection is not well defined in the context of type I IFN signalling. Here we show that HSV-2 replication was significantly higher in mice deficient in the type I IFN receptor or NK cells compared to wild type controls. There was no detectable IFN-γ production in the genital washes from IFN-α/βR(-/-) mice or NK cell depleted mice in response to HSV-2 infection compared to control mice. Absence of the type I IFN receptor does not alter homing of NK cells to the genital mucosa. Moreover, the absence of IL-12 had no significant effect on NK cell-derived IFN-γ. Surprisingly, IFN-α/βR(-/-) mice had more IL-15 positive cells in the genital mucosa in response to HSV-2 infection compared to control mice. We then examined the expression of IL-15 receptors on NK cells. There was no significant differences in the levels of IL-15 receptor expression on NK cells from IFN-α/βR(-/-) or control mice. Our data clearly suggest that type I IFN receptor signalling is essential for NK cell activation in response to genital HSV-2 infection, and propose that NK cell activation by IL-15 may involve type I IFNs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Genital epithelial cells (GECs) are the first line of mucosal defense against sexually transmitted infections. We exploited the ability of GECs to mount innate immune responses, by using TLR ligands to induce anti-viral activity against Herpes simplex virus, type 2 (HSV-2). Primary cultures of GECs were grown to confluent, polarized monolayers and found to express different levels of mRNA for TLR1-10. Innate anti-viral responses against HSV-2 infection were determined following treatment with eight different TLR ligands. HSV-2 replication was significantly inhibited following treatment with ligands for TLR3, 5 and 9, while lipo-polysaccharide (LPS), a TLR4 ligand, failed to provide any protection. Biologically active interferon-beta and nitric oxide production by GECs correlated with anti-viral activity. Following treatment with TLR3 ligand Poly I:C, inflammatory cytokines were upregulated. Poly I:C treatment led to activation of downstream transcription factors including interferon regulatory factor-3 (IRF-3) and NFkappaB. Anti-viral responses induced by TLR ligands in GECs may provide a unique alternative to topical microbicides by enhancing body's own mucosal innate defense mechanisms against sexually transmitted viruses.

Interleukin-15 (IL-15) is a cytokine important for the development, maturation, and function of many cells of the immune system including NK, NKT, gammadeltaT, and CD8(+) T cells. The relationship between IL-15 and B lymphocytes however, is not well characterized and is the focus of our study. Previous in vitro reports have shown that IL-15 increases proliferation of B lymphocytes and increases antibody secretion however, this relationship remains inadequately defined in vivo. The focus of this study was to examine the role of IL-15 in B cell homeostasis and function in vivo using mice that either over express IL-15 (IL-15tg mice) or are deficient in IL-15 (IL-15(-/-) mice) production. Here we report significant differences between the B cell populations of IL-15(-/-), C57BL/6, and IL-15tg mice. In fact, increased expression of IL-15 resulted in a significant decrease in the percentage and absolute number of CD19(+) cells. In vitro B cell co-cultures implicate interferon-gamma (IFN-gamma) as the factor responsible for inhibiting B cell proliferation. We also show that IL-15 expression affects B cell function, as B cells from IL-15 transgenic mice produce greater amounts of IgG and IgA than IL-15 knockout mice in vitro. Interestingly, despite significant differences in B cell numbers in these strains, there were no significant differences in total antibody titers in serum and vaginal washes of these mice. Results from our in vivo and in vitro experiments suggest that altered expression of IL-15 affects B cell homeostasis through the induction of NK cell-derived IFN-gamma.

Toll-like receptor (TLR) ligands are potent inducers of the innate immune system, of which NK and NKT cells play an important role. We examined the direct activation of highly purified human NK and/or NKT cells with known TLR ligands. NK/NKT cells were positive for all known TLR mRNA (TLR1-10). Ligands for TLR2-5 induced production of significant amounts of IFN-gamma by purified NK cells. However, a TLR9 ligand failed to induce significant levels of the cytokine. NK cells were depleted from PBMCs to confirm that they were the main source of IFN-gamma following treatment with TLR ligands, which resulted in a significant decrease in cytokines. The direct effects of TLR ligands on NK cytotoxicity were determined using 51Cr-labeled K562 target cells. Ligands for TLR2-5 were potent inducers of NK cell cytotoxicity, a TLR9 ligand was not. Our results suggest that TLR ligands can directly stimulate and enhance NK cell cytokine production and induce cytotoxic activities.

Mice genetically deficient in the natural killer (NK) cell lineage lack uterine (uNK cells) and demonstrate morphometrically-quantifiable histopathology within their implantation sites. Two particular mouse strains, tg(epsilon),26 and RAG-2 null x gamma(c) null, have been used successfully as transplant recipients to address questions relating to the biology of uNK cells. uNK cells did not differentiate within decidualized uterine graft segments from normal mice, which were anastomosed orthotopically into immunodeficient hosts. uNK cells did appear in similar grafts placed into immunocompetent hosts, indicating that uNK cells or their progenitors must home to the uterus. This was confirmed by splenocyte transplantation into pregnant uNK cell deficient recipients. Only splenocytes from pregnant donors, not those from non-pregnant donors, homed to the uterus. Homing in this in vivo assay was independent of the CC-chemokine receptors, CCR-2 and CCR-5. Longer-term bone marrow cell reconstitution of neonatal or virgin adult uNK cell-deficient mice has identified a functional role for uNK cells in modification of the decidual arterioles which is mediated by IFN-gamma. By utilizing mutant and gene-ablated mice as donors for tissue or haematopoietic cell transplants to uNK cell deficient mice, it should be possible to fully characterize the in vivo regulation and functions of these pregnancy-specific uterine lymphocytes.

These studies aimed to advance understanding of the functions of pregnancy-associated uterine lymphocytes of the natural killer (NK) cell lineage. The approach was morphometric analysis of implantation sites from timed pregnancies in genetically modified mice deficient in NK cells or in signaling associated with their major product, the cytokine interferon-gamma. In four different strains of pregnant, NK cell--deficient mice, the major decidual arterioles failed to undergo modifications to their smooth-muscle coats and displayed endothelial cell damage. Decidua lacked normal cell density. This pathology was observed by the end of the first trimester, before placental differentiation. By midgestation in these strains, placentas were smaller than in control strains. In normal mice, many uterine NK cells are perivascular in location and appear to be activated because they are the major sources of interferon-gamma and of the interferon-gamma--regulated enzyme inducible nitric oxide synthase. During pregnancy in mice genetically ablated for interferon-gamma, the interferon-gamma receptor chain-alpha or the transcription factor interferon regulatory factor-1, uterine NK cells differentiate but appear to be abnormal both morphologically and functionally. In these three strains, failure of pregnancy-induced vascular modifications and overt necrosis of decidua occur. Thus, in mice, lymphocytes of the NK cell lineage make specialized contributions to pregnancy-associated modification of the uterine vasculature and to maintenance of decidua. These contributions are achieved through interferon-gamma--mediated gene regulation and appear to enhance subsequent placental growth. Human CD56 bright decidual lymphocytes may have analogous functions. If so, changes in numbers or levels of activity of human uterine NK cells or mutations in genes regulated by uterine interferon-gamma could contribute to initiation of preeclampsia.

The efficacy of using human interleukin 8 (huIL-8) as an agent for inducing cervical relaxation in estrous and diestrous sheep was assessed in a small pilot study. Multiparous, estrus-synchronized ewes were treated for either 2 or 5 consecutive days with vaginal suppositories with or without 5 micrograms cytokine. Cervical penetration with an insemination instrument was then assessed in vivo. After euthanasia, physical, histological and enzymological properties of the cervix were examined. Treatment of diestrous sheep with huIL-8 did not result in recruitment of neutrophils into the cervix. Treatment of estrous sheep with huIL-8 usually led to neutrophil recruitment to the cervix and to either full or partial penetration of the cervix. However, some animals receiving placebo treatment had neutrophil infiltration of both the vagina and cervix and, in one of these, partial penetration of the cervix was also achieved. Thus, treatment with IL-8 as the sole agent in the vaginal suppository was not sufficient to relax the cervix of the nonpregnant ewe in this study.