Innate immune responses to human rotavirus in the neonatal gnotobiotic piglet disease model

Intestinal and systemic dendritic cell (DC) frequencies, serum and small intestinal content cytokines and uptake/binding of human rotavirus (HRV) virus‐like particles (VLP) were studied in HRV acutely infected or mock‐inoculated neonatal gnotobiotic piglets. Intestinal, mesenteric lymph node (MLN) and splenic plasmacytoid DCs (pDCs), conventional DCs (cDCs) and macrophages/monocytes were assessed by flow cytometry. In infected pigs, serum and small intestinal content interferon‐α (IFN‐α) were highest, interleukin‐12 (IL‐12) was lower and IL‐10, tumour necrosis factor‐α and IL‐6 were minimal. Compared with mock‐inoculated piglets, frequencies of total intestinal DCs were higher; splenic and MLN DC frequencies were lower. Most intestinal pDCs, but few cDCs, were IFN‐α⁺ and intestinal macrophages/monocytes were negative for IFN‐α. Serum IFN‐α levels and IFN‐α⁺ intestinal pDCs were highly correlated, suggesting IFN‐α production in vivo by intestinal pDCs (r = 0·8; P < 0·01). The intestinal pDCs and cDCs, but not intestinal macrophages/monocytes, of HRV‐infected piglets showed significantly lower VLP uptake/binding compared with mock‐inoculated piglets, suggesting higher activation of pDCs and cDCs in infected piglets. Both intestinal pDCs and cDCs were activated (IFN‐α⁺ and lower VLP binding) after HRV infection, suggesting their role in induction of HRV‐specific immunity. Dose‐effects of HRV on serum IFN‐α and IFN‐α⁺ DCs were studied by infecting piglets with 100‐fold higher HRV dose. A high dose increased parameters associated with inflammation (diarrhoea, intestinal pathology) but serum IFN‐α and IFN‐α⁺ DCs were similar between both groups. The pDCs have both anti‐ and pro‐inflammatory functions. Stimulation of the anti‐inflammatory effects of pDCs after the high dose, without increasing their pro‐inflammatory impacts, may be critical to reduce further immunopathology during HRV infection.     

Selective effects of Lactobacillus casei Shirota on T cell activation, natural killer cell activity and cytokine production

Modulation of host immunity is an important potential mechanism by which probiotics confer health benefits. This study was designed to investigate the effects of a probiotic strain, Lactobacillus casei Shirota (LcS), on immune function using human peripheral blood mononuclear cells (PBMC) in vitro. In addition, the role of monocytes in LcS‐induced immunity was also explored. LcS promoted natural killer (NK) cell activity and preferentially induced expression of CD69 and CD25 on CD8 ⁺ and CD56 ⁺ subsets in the absence of any other stimulus. LcS also induced production of interleukin (IL)‐1β, IL‐6, tumour necrosis factor (TNF)‐α, IL‐12 and IL‐10 in the absence of lipopolysaccharide (LPS). In the presence of LPS, LcS enhanced IL‐1β production but inhibited LPS‐induced IL‐10 and IL‐6 production, and had no further effect on TNF‐α and IL‐12 production. Monocyte depletion reduced significantly the impact of LcS on lymphocyte activation, cytokine production and natural killer (NK) cell activity. In conclusion, LcS activated cytotoxic lymphocytes preferentially in both the innate and specific immune systems, which suggests that LcS could potentiate the destruction of infected cells in the body. LcS also induced both proinflammatory and anti‐inflammatory cytokine production in the absence of LPS, but in some cases inhibited LPS‐induced cytokine production. Monocytes play an important role in LcS‐induced immunological responses.     

Effects of Lactobacillus GG treatment during pregnancy on the development of fetal antigen‐specific immune responses

Background Several clinical trials suggest that probiotics may have a role in the prevention of eczema. The optimal timing and mechanisms underlying this intervention are not clear. In particular it is not known whether such treatment works during pregnancy or whether postnatal exposure is important. Objective We investigated whether the probiotic Lactobacillus rhamnosus strain GG (LGG) influences fetal immune responses when administered to pregnant women, as a possible mechanism for its protective effects against the development of eczema. Methods Peripheral blood mononuclear cell from 11 adults treated with LGG, and cord blood mononuclear cells (CBMCs) from 73 women participating in a randomized controlled trial of LGG treatment were cultured with heat‐killed LGG, ovalbumin (OVA) or without stimulus. Cells were analysed by flow cytometry and real‐time PCR for markers of dendritic cell (DC) phenotype, T cell proliferation and regulation. Cytokine secretion was analysed in culture supernatants by multiplex cytokine assay. Results LGG treatment of adults led to systemic immune responses suggestive of antigen‐specific tolerance including reduced CD4⁺ T cell proliferation to heat‐killed LGG (30% reduction; P=0.03). LGG treatment of pregnant women did not influence CD4⁺ T cell proliferation, forkhead box P3 expression, DC phenotype or cytokine secretion in CBMCs cultured with heat‐killed LGG or OVA. Conclusion LGG treatment of pregnant women fails to influence fetal antigen‐specific immune responses. This suggests that modulation of fetal immune responses may not be a major mechanism by which probiotics such as LGG prevent eczema.     

DC therapy induces long‐term NK reactivity to tumors via host DC

We vaccinated mice with DC loaded with or without invariant NKT‐cell ligand α‐galactosylceramide and evaluated long‐term resistance against tumor challenge. When mice had been given either DC or DC/galactosylceramide and were challenged with tumor cells even 6–12 months later, both NK and NKT cells were quickly activated to express CD69 and produce IFN‐γ. The NK cells could resist a challenge with several different tumors in vivo. The activated NK and NKT cells could be depleted with anti‐NK1.1 treatment. In spite of this, the activated cells recovered, indicating that tumor‐responsive NK and NKT cells were being generated continuously as a result of vaccination with DC and were not true memory cells. The NK and NKT antitumor response in DC‐vaccinated mice depended on CD4⁺ T cells, but neither CD8⁺T cells nor CD4⁺CD25⁺ regulatory T cells. However, both vaccine DC and host DC were required for the development of long‐term, tumor reactive innate immunity. These results indicate that DC therapy in mice induces long‐lasting innate NK‐ and NKT‐cell activation through a pathway that requires host DC and CD4⁺ T cells and that the continued generation of active NK cells resists the establishment of metastases in vivo.     

IL‐33 promotes innate IFN‐γ production and modulates dendritic cell response in LCMV‐induced hepatitis in mice

Recent studies have revealed IL‐33 as a key factor in promoting antiviral T‐cell responses. However, it is less clear as to how IL‐33 regulates innate immunity. In this study, we infected wild‐type (WT) and IL‐33^?/? mice with lymphocytic choriomeningitis virus and demonstrated an essential role of infection‐induced IL‐33 expression for robust innate IFN‐γ production in the liver. We first show that IL‐33 deficiency resulted in a marked reduction in the number of IFN‐γ⁺ γδ T and NK cells, but an increase in that of IL‐17⁺ γδ T cells at 16 h postinfection. Recombinant IL‐33 (rIL‐33) treatment could reverse such deficiency via increasing IFN‐γ‐producing γδ T and NK cells, and inhibiting IL‐17⁺ γδ T cells. We also found that rIL‐33‐induced type 2 innate lymphoid cells were not involved in T‐cell responses and liver injury, since the adoptive transfer of type 2 innate lymphoid cells neither affected the IFN‐γ and TNF‐α production in T cells, nor liver transferase levels in lymphocytic choriomeningitis virus infected mice. Interestingly, we found that while IL‐33 was not required for costimulatory molecule expression, it was critical for DC proliferation and cytokine production. Together, this study highlights an essential role of IL‐33 in regulating innate IFN‐γ‐production and DC function during viral hepatitis.     

Innate and adaptive stimulation of murine diverse NKT cells result in distinct cellular responses

Natural killer T (NKT) cells recognize glycolipids presented on CD1d. They share features of adaptive T lymphocytes and innate NK cells, and mediate immunoregulatory functions via rapid production of cytokines. Invariant (iNKT) and diverse (dNKT) NKT cell subsets are defined by their TCR. The immunological role of dNKT cells, that do not express the invariant TCRα‐chain used by iNKT cells, is less well explored than that of iNKT cells. Here, we investigated signals driving Toll‐like receptor (TLR) ligand activation of TCR‐transgenic murine dNKT cells. IFN‐γ production by dNKT cells required dendritic cells (DC), cell‐to‐cell contact and presence of TLR ligands. TLR‐stimulated DC activated dNKT cells to secrete IFN‐γ in a CD1d‐, CD80/86‐ and type I IFN‐independent manner. In contrast, a requirement for IL‐12p40, and a TLR ligand‐selective dependence on IL‐18 or IL‐15 was observed. TLR ligand/DC stimulation provoked early secretion of pro‐inflammatory cytokines by both CD62L⁺ and CD62L^? dNKT cells. However, proliferation was limited. In contrast, TCR/co‐receptor‐mediated activation resulted in proliferation and delayed production of a broader cytokine spectrum preferentially in CD62L^? dNKT cells. Thus, innate (TLR ligand/DC) and adaptive (TCR/co‐receptor) stimulation of dNKT cells resulted in distinct cellular responses that may contribute differently to the formation of immune memory.     

Lactobacillus GG has in vitro effects on enhanced interleukin-10 and interferon-γ release of mononuclear cells but no in vivo effects in supplemented mothers and their neonates

Background The value of probiotics for primary prevention is controversial. Moreover, only little is known about the underlying immunological mechanisms of action. Therefore, we assessed the proliferative response and cytokine release in cultures of isolated mononuclear cells from pregnant women and their neonates supplemented with Lactobacillus GG (LGG) or placebo. Methods In a double‐blind, placebo‐controlled prospective trial, pregnant women with at least one first‐degree relative or a partner with an atopic disease were randomly assigned to receive either the probiotic LGG (ATCC 53103; 5 × 10⁹ colony‐forming units LGG twice daily) or placebo 4–6 weeks before expected delivery, followed by a post‐natal period of 6 months. Cord blood mononuclear cells (CBMC) and peripheral blood mononuclear cells (PBMC) of the corresponding mother were isolated from cord blood and peripheral blood (n=68). The proliferative response of CBMC and PBMC was expressed as the stimulation index (SI), which was calculated according to the ratio between the mean counts per minute (c.p.m.) values measured in the wells with stimulated cells and the mean c.p.m. values measured in the wells with unstimulated cells. Additionally, the cytokines IFN‐γ, IL‐10 and IL‐13 in the cell culture supernatants were measured using the ELISA technique. Results No difference was observed between the LGG‐supplemented group and the placebo group in terms of the proliferative capacity of maternal or neonatal cord blood cells in response to IL‐2, β‐lactoglobulin or LGG. In vitro stimulation with LGG resulted in significantly enhanced release of IL‐10 and IFN‐γ, compared with cytokine release in unstimulated controls. However, this phenomenon was observed in supernatants of maternal and neonatal MC in both groups, independent of prior supplementation with LGG. Conclusion LGG has in vitro effects on enhanced IL‐10 and IFN‐γ release of mononuclear cells. However, supplementation with LGG during pregnancy did not alter the proliferative capacity or cytokine pattern in their recipients.     

Innate,antigen‐independent role for T cells in the activation of the immune system by Propionibacterium acnes

Propionibacterium acnes is a human commensal but also an opportunistic pathogen. In mice, P. acnes exerts strong immunomodulatory activities, including formation of intrahepatic granulomas and induction of LPS hypersensitivity. These activities are dependent on P. acnes recognition via TLR9 and subsequent IL‐12‐mediated IFN‐γ production. We show that P. acnes elicits IL‐12p40 and p35 mRNA expression in macrophages, and IFN‐γ mRNA in liver CD4⁺ T cells and NK cells. After priming with P. acnes, CD4⁺ T cells serve as the major IFN‐γ mRNA source. In the absence of CD4⁺ T cells, CD8⁺ T cells (regardless of antigenic specificity) or NK cells can produce sufficient IFN‐γ to induce the P. acnes‐driven immune effects. Moreover, in the absence of αβT cells, γδT cells also enable the development of strongly enhanced TNF‐α and IFN‐γ responses to LPS and intrahepatic granuloma formation. Thus, under microbial pressure, different T‐cell types, independent of their antigen specificity, exert NK‐cell‐like functions, which contribute decisively to the activation of the innate immune system.     

Probiotic Escherichia coli Nissle 1917 activates DC and prevents house dust mite allergy through a TLR4‐dependent pathway

Experimental animal and human studies have demonstrated that probiotic strains have beneficial effects on allergy. Here we report that the probiotic Escherichia coli Nissle 1917 strain (EcN) is able to activate DC, as shown by important cytokine synthesis together with up‐regulation of membrane expression of CD40, CD80 and CD86. This EcN‐induced DC activation was strictly dependent on the TLR4 signaling pathway and was also associated with stimulation of NF‐κB and MAPK. We next investigated the prophylactic potential of i.n. co‐administration of EcN with a recombinant form of Der p 1 (ProDer p 1) in a murine model of mite allergy. I.n. vaccinations with EcN plus ProDer p 1 prevented the subsequent allergic response following Der p 1 sensitization and airway challenge with aerosolized mite extracts through the induction of an allergen‐specific IgG2a response, the prevention of specific IgE production and a strong reduction of IL‐5 secretion by allergen‐restimulated splenocytes. EcN alone or in combination with ProDer p 1 inhibited the development of airway eosinophilia and neutrophilia. This in vivo protective effect of EcN was, in part, mediated by TLR4 signaling. Our results suggest that EcN represents an efficient adjuvant to prevent allergic responses.     

Natural killer and dendritic cells collaborate in the immune response induced by the vaccine against uterine cervical cancer

Virus‐like particles (VLPs) of human papillomavirus (HPV) are used as a vaccine against HPV‐induced cancer, and recently we have shown that these VLPs are able to activate natural killer (NK) cells. Since NK cells collaborate with dendritic cells (DCs) to induce an immune response against viral infections and tumors, we studied the impact of this crosstalk in the context of HPV vaccination. NK cells in the presence of HPV‐VLPs enhanced DC‐maturation as shown by an upregulation of CD86 and HLA‐DR and an increased production of IL‐12p70, but not of the immunosuppressive cytokine IL‐10. This activation was bidirectional. Indeed, in the presence of HPV‐VLPs, DCs further activated NK cells by inducing the upregulation of cell surface activation markers (CD69 and HLA‐DR). The function of NK cells was also improved as shown by an increase in IFN‐γ secretion and cytotoxic activity against an HPV⁺ cell line. This crosstalk between NK cells and DCs needed CD40 interaction and IL‐12p70 secretion, whereas NKG2D was not implicated. Our results provide insight into how VLPs interact with innate immune cells and how NK cells and DCs play a role in the immune response induced by this vaccine agent.     

Differential recruitment and activation of natural killer cell sub‐populations by Mycobacterium bovis‐infected dendritic cells

Through complex interplay with APCs, subsets of NK cells play an important role in shaping adaptive immune responses. Bovine tuberculosis, caused by Mycobacterium bovis, is increasing in incidence and detailed knowledge of host–pathogen interactions in the natural host is essential to facilitate disease control. We investigated the interactions of NK‐cell sub‐populations and M. bovis‐infected DCs to determine early innate mechanisms in the response to infection. A sub‐population of NK cells (NKp46⁺CD2^?) selectively expressing lymphoid homing and inflammatory chemokine receptors were induced to migrate towards M. bovis‐infected DCs. This migration was associated with increased expression of chemokines CCL3, 4, 5, 20 and CXCL8 by M. bovis‐infected DCs. Activation of NKp46⁺CD2^? NK cells and secretion of IFN‐γ was observed, a response reliant on localised IL‐12 release and direct cellular interaction. In a reciprocal manner, NKp46⁺CD2^? cells induced an increase in the intensity of cell surface MHC class II expression on DCs. In contrast, NKp46⁺CD2⁺ NK cells were unable to secrete IFN‐γ and did not reciprocally affect DCs. This study provides novel evidence to demonstrate distinct effector responses between bovine NK‐cell subsets during mycobacterial infection.     

NK cells modulate the lung dendritic cell‐mediated Th1/Th17 immunity during intracellular bacterial infection

The impact of the interaction between NK cells and lung dendritic cells (LDCs) on the outcome of respiratory infections is poorly understood. In this study, we investigated the effect and mechanism of NK cells on the function of LDCs during intracellular bacterial lung infection of Chlamydia muridarum in mice. We found that the naive mice receiving LDCs from C. muridarum‐infected NK‐cell‐depleted mice (NK‐LDCs) showed more serious body weight loss, bacterial burden, and pathology upon chlamydial challenge when compared with the recipients of LDCs from infected sham‐treated mice (NK+LDCs). Cytokine analysis of the local tissues of the former compared with the latter exhibited lower levels of Th1 (IFN‐γ) and Th17 (IL‐17), but higher levels of Th2 (IL‐4), cytokines. Consistently, NK‐LDCs were less efficient in directing C. muridarum‐specific Th1 and Th17 responses than NK+LDCs when cocultured with CD4⁺ T cells. In NK cell/LDC coculture experiments, the blockade of NKG2D receptor reduced the production of IL‐12p70, IL‐6, and IL‐23 by LDCs. The neutralization of IFN‐γ in the culture decreased the production of IL‐12p70 by LDCs, whereas the blockade of TNF‐α resulted in diminished IL‐6 production. Our findings demonstrate that NK cells modulate LDC function to elicit Th1/Th17 immunity during intracellular bacterial infection.     

IL‐18‐induced expression of high‐affinity IL‐2R on murine NK cells is essential for NK‐cell IFN‐γ production during murine Plasmodium yoelii infection

Early production of pro‐inflammatory cytokines, including IFN‐γ, is essential for control of blood‐stage malaria infections. We have shown that IFN‐γ production can be induced among human natural killer (NK) cells by coculture with Plasmodium falciparum infected erythrocytes, but the importance of this response is unclear. To further explore the role of NK cells during malaria infection, we have characterized the NK‐cell response of C57BL/6 mice during lethal (PyYM) or nonlethal (Py17XNL) P. yoelii infection. Ex vivo flow cytometry revealed that NK cells are activated within 24 h of Py17XNL blood‐stage infection, expressing CD25 and producing IFN‐γ; this response was blunted and delayed during PyYM infection. CD25 expression and IFN‐γ production were highly correlated, suggesting a causal relationship between the two responses. Subsequent in vitro experiments revealed that IL‐18 signaling is essential for induction of CD25 and synergizes with IL‐12 to enhance CD25 expression on splenic NK cells. In accordance with this, Py17XNL‐infected erythrocytes induced NK‐cell CD25 expression and IFN‐γ production in a manner that is completely IL‐18‐ and partially IL‐12‐dependent, and IFN‐γ production is enhanced by IL‐2. These data suggest that IL‐2 signaling via CD25 amplifies IL‐18‐ and IL‐12‐mediated NK‐cell activation during malaria infection.     

DC within the pregnant mouse uterus influence growth and functional properties of uterine NK cells

The vascular addressins mucosal addressin cell adhesion molecule‐1, P‐selectin and ICAM‐1 permit α₄β₇‐integrin‐expressing DC, especially those of the myeloid lineage (CD11c⁺CD11b⁺ DC), to access the pregnant mouse uterus. Injection of blocking monoclonal antibodies against mucosal addressin cell adhesion molecule‐1 in P‐selectin^?/? mice or experimental approaches with β7‐integrin^?/? or ICAM‐1^?/? mice revealed that limited access or absence of CD11c⁺CD11b⁺ DC at the maternal/fetal interface negatively affects the frequency, size and functional properties of uterine NK (uNK) cells. Adoptive transfer of DC obtained from WT mice into β7‐integrin^?/? mice abrogates these effects and emphasizes the importance of DC in uNK cell differentiation. Interestingly, those implantation sites lacking CD11c⁺CD11b⁺ DC are characterized by decreased IL‐15 and IL‐12 mRNA and/or protein levels. Chronic administration of IL‐15 in these mice gives rise to uNK cell numbers and size comparable to those of WT mice, whereas additional injection of IL‐12 positively affects the IFN‐γ expression of uNK cells. Real‐time RT‐PCR and protein arrays performed with isolated uterine DC underline the role of DC as a source of IL‐15 and IL‐12 in the pregnant mouse uterus.     

Control of Mycobacterium tuberculosis growth by activated natural killer cells

We characterized the underlying mechanisms by which glutathione (GSH)‐enhanced natural killer (NK) cells inhibit the growth of Mycobacterium tuberculosis (M. tb) inside human monocytes. We observed that in healthy individuals, treatment of NK cells with N‐acetyl cysteine (NAC), a GSH prodrug in conjunction with cytokines such as interleukin (IL)‐2 + IL‐12, resulted in enhanced expression of NK cytotoxic ligands (FasL and CD40L) with concomitant stasis in the intracellular growth of M. tb. Neutralization of FasL and CD40L in IL‐2 + IL‐12 + NAC‐treated NK cells resulted in abrogation in the growth inhibition of M. tb inside monocytes. Importantly, we observed that the levels of GSH are decreased significantly in NK cells derived from individuals with HIV infection compared to healthy subjects, and this decrease correlated with a several‐fold increase in the growth of M. tb inside monocytes. This study describes a novel innate defence mechanism adopted by NK cells to control M. tb infection.     

Protective dendritic cell responses against listeriosis induced by the short form of the deubiquitinating enzyme CYLD are inhibited by full‐length CYLD

The deubiquitinating enzyme CYLD is an important tumor suppressor and inhibitor of immune responses. In contrast to full‐length CYLD, the immunological function of the naturally occurring short splice variant of CYLD (sCYLD) is insufficiently described. Previously, we showed that DCs, which lack full‐length CYLD but express sCYLD, exhibit augmented NF‐κB and DC activation. To explore the function of sCYLD in infection, we investigated whether DC‐specific sCYLD regulates the pathogenesis of listeriosis. Upon Listeria monocytogenes infection of CD11c‐Cre Cyld^{ex7/8 fl/fl} mice, infection of CD8α⁺ DCs, which are crucial for the establishment of listeriosis in the spleen, was not affected. However, NF‐κB activity of CD11c‐Cre Cyld^{ex7/8 fl/fl} DCs was increased, while activation of ERK and p38 was normal. In addition, CD11c‐Cre Cyld^{ex7/8 fl/fl} DCs produced more TNF, IL‐10, and IL‐12 upon infection, which led to enhanced stimulation of IFN‐γ‐producing NK cells. In addition CD11c‐Cre Cyld^{ex7/8 fl/fl} DCs presented Listeria Ag more efficiently to CD8⁺ T cells resulting in a stronger pathogen‐specific CD8⁺ T‐cell proliferation and more IFN‐γ production. Collectively, the improved innate and adaptive immunity and survival during listeriosis identify the DC‐specific FL‐CYLD/sCYLD balance as a potential target to modulate NK‐cell and Ag‐specific CD8⁺ T‐cell responses.     

Deficient arginase II expression without alteration in arginase I expression attenuated experimental autoimmune encephalomyelitis in mice

In the past there have been a multitude of studies that ardently support the role of arginase II (Arg II) in vascular and endothelial disorders; however, the regulation and function of Arg II in autoimmune diseases has thus far remained unclear. Here we report that a global Arg II null mutation in mice suppressed experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. During EAE, both Arg I and Arg II were induced in spinal cords, but only Arg II was induced in spleens and splenic dendritic cells (DCs). DC activation by lipopolysaccharide (LPS), CD40L or TLR8 agonist significantly enhanced Arg II expression without affecting Arg I expression. Conversely, DC differentiating cytokines [IL‐4 and granulocyte macrophage‐colony‐stimulating factor (GM‐CSF)] yielded opposite effects. In addition, Arg I and Arg II were regulated differentially during Th1 and Th17 cell polarization. Arg II deficiency in mice delayed EAE onset, ameliorated clinical symptoms and reduced myelin loss, accompanied by a remarkable reduction in the EAE‐induced spinal cord expression of Th17 cell markers (IL‐17 and RORγt). The abundance of Th17 cells and IL‐23⁺ cells in relevant draining lymph nodes was significantly reduced in Arg II knockout mice. In activated DCs, Arg II deficiency significantly suppressed the expression of Th17‐differentiating cytokines IL‐23 and IL‐6. Interestingly, Arg II deficiency did not lead to any compensatory increase in Arg I expression in vivo and in vitro. In conclusion, Arg II was identified as a factor promoting EAE likely via an Arg I‐independent mechanism. Arg II may promote EAE by enhancing DC production of Th17‐differentiating cytokines. Specific inhibition of Arg II could be a potential therapy for multiple sclerosis.