Histamine inhibits the production of interleukin-12 through interaction with H2 receptors.

IL-12 is essential for T helper 1 (Th1) development and inhibits the induction of Th2 responses. Atopic diseases, which are characterized by Th2 responses, are associated with the overproduction of histamine. Here we present evidence that histamine, at physiological concentrations, strongly inhibits human IL-12 p40 and p70 mRNA and protein production by human monocytes. The use of specific histamine receptor antagonists reveals that this inhibition is mediated via the H2 receptor and induction of intracellular cAMP. The inhibition of IL-12 production is independent of IL-10 and IFN-gamma. The observation that histamine strongly reduces the production of the Th1-inducing cytokine IL-12 implies a positive feedback mechanism for the development of Th2 responses in atopic patients.     

IL-18 gene therapy develops Th1-type immune responses in Leishmania major-infected BALB/c mice: is the effect mediated by the CpG signaling TLR9?

IL-18 regulates either Th1 or Th2 responses depending on the cytokine microenvironment. Administration of recombinant IL-18 (rIL-18) alone does not promote Th1 response, but rather induces Th2 response and exacerbates Leishmania major infection in susceptible BALB/c mice. Here, we treated BALB/c mice with an IL-18-expressing plasmid by using a gene gun weekly after L. major infection. This gene therapy resulted in improved pathogenic process and preferential induction of Th1 responses by inducing the expression of IL-12 p40, but treatment with rIL-18 did not. Notably, simultaneous administration of rIL-18 with an empty plasmid vector rendered BALB/c mice resistant to the infection, despite the fact that treatment with either rIL-18 alone or the plasmid vector alone did not influence the susceptibility. The synergistic role of the vector with rIL-18 was found to depend on CpG motifs, which enhanced expression of proinflammatory cytokines, especially IL-12, from APCs through Toll-like receptor (TLR) 9 ligation. Treatment with methylated plasmid vector in which CpG was disrupted could no longer prevent the disease development in coadministration with rIL-18. Taken together, IL-18 gene therapy was shown to develop Th1-type protective immunity in L. major-infected BALB/c mice without the requirement of exogenous IL-12, probably via CpG-TLR9 signaling pathway.     

Experimental autoimmune encephalitis and inflammation in the absence of interleukin-12

IL-12 is considered a critical proinflammatory cytokine for autoimmune diseases such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). IL-12 is a heterodimer composed of a p35 subunit and a common p40 subunit shared by other cytokines. Both IL-12 p40(-/-) and p35(-/-) mice fail to produce IL-12 p70 heterodimer. However, in contrast to p40(-/-) mice, p35(-/-) mice are highly susceptible to the induction of EAE, establishing that IL-12 p70 is not essential for the development of EAE. When compared with wild-type mice, both p40(-/-) and p35(-/-) mice show deficiencies in primary IFN-gamma responses by lymph node cells. Expression profiling of the inflamed CNS revealed that Th2 cytokines such as IL-4 and IL-10 are upregulated in p35(-/-) mice, whereas LT-alpha and TNF-alpha levels are reduced. These studies show that a molecule other than IL-12 p70, which uses the p40 subunit, fulfills the functions previously attributed to IL-12 with regard to the development and pathogenesis of this autoimmune disease.     

Dectin-1 diversifies Aspergillus fumigatus-specific T cell responses by inhibiting T helper type 1 CD4 T cell differentiation

Pulmonary infection of mice with Aspergillus fumigatus induces concurrent T helper type 1 (Th1) and Th17 responses that depend on Toll-like receptor/MyD88 and Dectin-1, respectively. However, the mechanisms balancing Th1 and Th17 CD4 T cell populations during infection remain incompletely defined. In this study, we show that Dectin-1 deficiency disproportionally increases Th1 responses and decreases Th17 differentiation after A. fumigatus infection. Dectin-1 signaling in A. fumigatus-infected wild-type mice reduces IFN-γ and IL-12p40 expression in the lung, thereby decreasing T-bet expression in responding CD4 T cells and enhancing Th17 responses. Absence of IFN-γ or IL-12p35 in infected mice or T-bet in responding CD4 T cells enhances Th17 differentiation, independent of Dectin-1 expression, in A. fumigatus-infected mice. Transient deletion of monocyte-derived dendritic cells also reduces Th1 and boosts Th17 differentiation of A. fumigatus-specific CD4 T cells. Our findings indicate that Dectin-1-mediated signals alter CD4 T cell responses to fungal infection by decreasing the production of IL-12 and IFN-γ in innate cells, thereby decreasing T-bet expression in A. fumigatus-specific CD4 T cells and enabling Th17 differentiation.     

Polyubiquitin binding to ABIN1 is required to prevent autoimmunity

The protein ABIN1 possesses a polyubiquitin-binding domain homologous to that present in nuclear factor κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of NF-κB (IκB) kinase (IKK) complex. To address the physiological significance of polyubiquitin binding, we generated knockin mice expressing the ABIN1[D485N] mutant instead of the wild-type (WT) protein. These mice developed all the hallmarks of autoimmunity, including spontaneous formation of germinal centers, isotype switching, and production of autoreactive antibodies. Autoimmunity was suppressed by crossing to MyD88(-/-) mice, demonstrating that toll-like receptor (TLR)-MyD88 signaling pathways are needed for the phenotype to develop. The B cells and myeloid cells of the ABIN1[D485N] mice showed enhanced activation of the protein kinases TAK, IKK-α/β, c-Jun N-terminal kinases, and p38α mitogen-activated protein kinase and produced more IL-6 and IL-12 than WT. The mutant B cells also proliferated more rapidly in response to TLR ligands. Our results indicate that the interaction of ABIN1 with polyubiquitin is required to limit the activation of TLR-MyD88 pathways and prevent autoimmunity.     

Primary role for Gi protein signaling in the regulation of interleukin 12 production and the induction of T helper cell type 1 responses

We explored the role of Gi protein signaling in the regulation of interleukin (IL)-12 production and T helper cell type 1 (Th1) T cell differentiation. In initial studies, we showed that treatment of normal mice with pertussis toxin (PT), which inhibits Gi protein signaling, enhanced the capacity of splenocytes to produce IL-12 in response to both microbial and nonmicrobial stimuli. In addition, PT treatment increased the production of tumor necrosis factor (TNF)-alpha and IL-10 by stimulated cells. These findings were corroborated by the fact that untreated Gi2alpha(2/-) mice exhibited enhanced production of IL-12 and TNF-alpha by splenocytes, and of IL-12 p40 by purified spleen CD8alpha(+) lymphoid dendritic cells. Finally, we showed that while normal BALB/c mice infected with Leishmania major exhibited a nonhealing phenotype, those treated with PT when infection was initiated exhibited a healing phenotype along with an enhancement of leishmania-specific Th1 responses in draining lymph nodes. Further, healing was prevented by coadministration of anti-IL-12 and PT. These data demonstrate that endogenous Gi protein signaling has a primary role in the regulation of IL-12 production and the induction of Th1 responses in vivo.     

Eosinophil-derived neurotoxin acts as an alarmin to activate the TLR2-MyD88 signal pathway in dendritic cells and enhances Th2 immune responses

Eosinophil-derived neurotoxin (EDN) is an eosinophil granule-derived secretory protein with ribonuclease and antiviral activity. We have previously shown that EDN can induce the migration and maturation of dendritic cells (DCs). Here, we report that EDN can activate myeloid DCs by triggering the Toll-like receptor (TLR)2-myeloid differentiation factor 88 signaling pathway, thus establishing EDN as an endogenous ligand of TLR2. EDN activates TLR2 independently of TLR1 or TLR6. When mice were immunized with ovalbumin (OVA) together with EDN or with EDN-treated OVA-loaded DCs, EDN enhanced OVA-specific T helper (Th)2-biased immune responses as indicated by predominant production of OVA-specific interleukin (IL)-5, IL-6, IL-10, and IL-13, as well as higher levels of immunoglobulin (Ig)G1 than IgG2a. Based on its ability to serve as a chemoattractant and activator of DCs, as well as the capacity to enhance antigen-specific immune responses, we consider EDN to have the properties of an endogenous alarmin that alerts the adaptive immune system for preferential enhancement of antigen-specific Th2 immune responses.     

Toll-like receptor 4–mediated lymphocyte influx induces neonatal necrotizing enterocolitis

The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (Rag1^–/–) mice were protected from NEC and transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. The intestinal expression of the lipopolysaccharide receptor TLR4, which is higher in the premature compared with full-term human and mouse intestine, is required for lymphocyte influx through TLR4-mediated upregulation of CCR9/CCL25 signaling. TLR4 also mediates a STAT3-dependent polarization toward increased proinflammatory CD3⁺CD4⁺IL-17⁺ and reduced tolerogenic Foxp3⁺ Treg lymphocytes (Tregs). Th17 lymphocytes were required for NEC development, as inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, while IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC. Importantly, TLR4-dependent Th17 polarization could be reversed by the enteral administration of retinoic acid, which induced Tregs and decreased NEC severity. These findings identify an important role for proinflammatory lymphocytes in NEC development via intestinal epithelial TLR4 that could be reversed through dietary modification.     

Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells

IL-17 is implicated in the pathogenesis of rheumatoid arthritis (RA) and has previously been shown to be induced by tumor necrosis factor (TNF) in vitro. The aim of this study was to assess the impact of TNF inhibition on IL-17 production in collagen-induced arthritis, a model of RA. TNF blockade using TNFR-Fc fusion protein or anti-TNF monoclonal antibody reduced arthritis severity but, unexpectedly, expanded populations of Th1 and Th17 cells, which were shown by adoptive transfer to be pathogenic. Th1 and Th17 cell populations were also expanded in collagen-immunized TNFR p55^−/− but not p75^−/− mice. The expression of IL-12/IL-23 p40 was up-regulated in lymph nodes (LN) from p55^−/− mice, and the expansion of Th1/Th17 cells was abrogated by blockade of p40. Treatment of macrophages with rTNF also inhibited p40 production in vitro. These findings indicate that at least one of the ways in which TNF regulates Th1/Th17 responses in arthritis is by down-regulating the expression of p40. Finally, although TNF blockade increased numbers of Th1 and Th17 cells in LN, it inhibited their accumulation in the joint, thereby providing an explanation for the paradox that anti-TNF therapy ameliorates arthritis despite increasing numbers of pathogenic T cells.     

The protective role of TLR6 in a mouse model of asthma is mediated by IL-23 and IL-17A

TLRs are a family of receptors that mediate immune system pathogen recognition. In the respiratory system, TLR activation has both beneficial and deleterious effects in asthma. For example, clinical data indicate that TLR6 activation exerts protective effects in asthma. Here, we explored the mechanism or mechanisms through which TLR6 mediates this effect using mouse models of Aspergillus fumigatus-induced and house dust mite antigen-induced (HDM antigen-induced) chronic asthma. Tlr6-/- mice with fungal- or HDM antigen-induced asthma exhibited substantially increased airway hyperresponsiveness, inflammation, and remodeling compared with WT asthmatic groups. Surprisingly, whole-lung levels of IL-23 and IL-17 were markedly lower in Tlr6-/- versus WT asthmatic mice. Tlr6-/- DCs generated less IL-23 upon activation with lipopolysaccharide, zymosan, or curdlan. Impaired IL-23 generation in Tlr6-/- mice also corresponded with lower levels of expression of the pathogen-recognition receptor dectin-1 and expansion of Th17 cells both in vivo and in vitro. Exogenous IL-23 treatment of asthmatic Tlr6-/- mice restored IL-17A production and substantially reduced airway hyperresponsiveness, inflammation, and lung fungal burden compared with that in untreated asthmatic Tlr6-/- mice. Together, our data demonstrate that TLR6 activation is critical for IL-23 production and Th17 responses, which both regulate the allergic inflammatory response in chronic fungal-induced asthma. Thus, therapeutics targeting TLR6 activity might prove efficacious in the treatment of clinical asthma.     

Yellow fever vaccine YF-17D activates multiple dendritic cell subsets via TLR2, 7, 8, and 9 to stimulate polyvalent immunity

The live attenuated yellow fever vaccine 17D (YF-17D) is one of the most effective vaccines available, with a 65-yr history of use in >400 million people globally. Despite this efficacy, there is presently no information about the immunological mechanisms by which YF-17D acts. Here, we present data that suggest that YF-17D activates multiple Toll-like receptors (TLRs) on dendritic cells (DCs) to elicit a broad spectrum of innate and adaptive immune responses. Specifically, YF-17D activates multiple DC subsets via TLRs 2, 7, 8, and 9 to elicit the proinflammatory cytokines interleukin (IL)-12p40, IL-6, and interferon-alpha. Interestingly, the resulting adaptive immune responses are characterized by a mixed T helper cell (Th)1/Th2 cytokine profile and antigen-specific CD8+ T cells. Furthermore, distinct TLRs appear to differentially control the Th1/Th2 balance; thus, whilst MyD88-deficient mice show a profound impairment of Th1 cytokines, TLR2-deficient mice show greatly enhanced Th1 and Tc1 responses to YF-17D. Together, these data enhance our understanding of the molecular mechanism of action of YF-17D, and highlight the potential of vaccination strategies that use combinations of different TLR ligands to stimulate polyvalent immune responses.     

Role of MyD88 and TLR9 in the innate immune response elicited by serotype 5 adenoviral vectors

A replication-incompetent adenoviral (Ad) vector is generating interest for both gene therapy and immunotherapy. A major limitation of the use of Ad vectors is the innate immune response, which causes inflammatory cytokine production and tissue damage; however, the precise mechanism of the innate immune response remains to be clarified. Here, we show that serotype 5 human Ad vectors elicit innate immune responses through a myeloid differentiating factor 88 (MyD88)/Toll-like receptor (TLR)-9-dependent and/or -independent manner according to cell type. After stimulation with Ad vectors, the production of interleukin (IL)-6 and IL-12 was significantly decreased in MyD88- or TLR9-deficient dendritic cells (DCs), compared with wild-type DCs. In addition, the surface expression of maturation marker proteins, such as CD40, CD80, CD86, and MHC class II, in MyD88- or TLR9-deficient granulocyte-macrophage colony-stimulating factor (GM-CSF)-DCs was similar to that in wild-type DCs. On the other hand, MyD88- or TLR9-deficient peritoneal macrophages produced the same level of IL-6 as wild-type macrophages after infection with Ad vectors. We did not find any differences in the mRNA expression levels of the molecules involved in innate immunity, such as MyD88, TLR3, TLR7, and TLR9, between DCs and macrophages. The intravenous injection of luciferase-expressing Ad vectors into MyD88- or TLR9-deficient mice resulted in almost comparable levels of IL-6 and IL-12 production and luciferase expression with wild-type mice. These results suggest that Ad vectors can activate innate immunity via MyD88/TLR9-dependent and -independent mechanisms.     

Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6

Transition of immature antigen presenting cells (APCs) to the state of professional APCs is essential for initiation of cell-mediated immune responses to pathogens. Signal transduction via molecules of the Toll-like receptor (TLR)/interleukin 1 receptor (IL-1R) pathway is critical for activation of APCs either by pathogen-derived pattern ligands like lipopolysaccharides (LPS) or by CD40 ligation through T helper cells. The capacity of bacterial DNA (CpG-DNA) to induce APCs to differentiate into professional APCs represents an interesting discovery. However, the signaling pathways involved are poorly understood. Here we show that CpG-DNA activates the TLR/IL-1R signaling pathway via the molecules myeloid differentiation marker 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6), leading to activation of kinases of the IkappaB kinase complex and the c-jun NH(2)-terminal kinases. Moreover, cells of TLR2- and TLR4-deficient mice are activated by CpG-DNA, whereas cells of MyD88-deficient mice do not respond. The data suggest that CpG-DNA initiates signaling via the TLR/IL-1R pathway in APCs in a manner similar to LPS and to T helper cell-mediated CD40 ligation. Activation of the TLR/IL-1R signaling pathway by foreign bacterial DNA may be one way to initiate innate defense mechanisms against infectious pathogens in vivo.     

Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.     

Bias in macrophage activation pattern influences non-alcoholic steatohepatitis (NASH) in mice

In humans, there is large inter-individual variability in the evolution of NAFLD (non-alcoholic fatty liver disease) to NASH (non-alcoholic steatohepatitis). To investigate this issue, NASH was induced with an MCD (methionine-choline-deficient) diet in C57BL/6 and Balb/c mice that are characterized by different biases in Th1/Th2 and macrophage (M1/M2) responses. Following 4?weeks on the MCD diet, steatosis and lobular inflammation were prevalent in C57BL/6 (Th1, M1 oriented) than in Balb/c (Th2, M2 oriented) mice. Consistently, hepatic TNFα (tumour necrosis factor α) mRNA expression and circulating TNFα levels were higher in MCD-fed C57BL/6 than in MCD-fed Balb/c mice. The Th1/Th2 bias did not account for the increased NASH severity, as in both strains MCD feeding did not significantly modify the liver mRNA expression of the Th1 markers IFNγ (interferon γ) and T-bet or that of the Th2 markers IL-4 (interleukin 4) and GATA-3. Conversely, MCD-fed C57BL/6 mice displayed higher liver mRNAs for the macrophage M1 activation markers iNOS (inducible NO synthase), IL-12p40 and CXCL10 (CXC chemokine ligand 10) than similarly treated Balb/c mice, without effects on the M2 polarization markers IL-10 and MGL-1 (macrophage galactose-type C-type lectin-1). Circulating IL-12 was also higher in MCD-fed C57BL/6 than in MCD-fed Balb/c mice. The analysis of macrophages isolated from the livers of MCD-fed animals confirmed an enhanced expression of M1 markers in C57BL/6 mice. Among all of the MCD-treated mice, liver iNOS, IL-12p40 and CXCL10 mRNA levels positively correlated with the frequency of hepatic necro-inflammatory foci. We concluded that the macrophage M1 bias in C57BL/6 mice may account for the increased severity of NASH in this strain, suggesting macrophage responses as important contributors to NAFLD progression.     

Recombinant interleukin 12 cures mice infected with Leishmania major

Resistant C57BL/6 mice infected with Leishmania major are self-healing, whereas susceptible BALB/c mice fail to contain cutaneous infection and subsequently undergo fatal visceral dissemination. These disparate outcomes are mediated by dissimilar expansions of T helper type 1 (Th1) and Th2 CD4+ T lymphocyte subsets in vivo during cure and progression of disease. Because interleukin 12 (IL-12) has potent T cell growth and interferon gamma (IFN-gamma) stimulatory effects, we studied its effect on CD4+ T cell differentiation during murine leishmaniasis. Treatment with recombinant murine (rMu)IL-12 during the first week of infection cured 89% of normally susceptible BALB/c mice, as defined by decreased size of infected footpads and 1,000-10,000-fold reduced parasite burdens, and provided durable resistance against reinfection. Cure was associated with markedly depressed production of IL-4 by lymph node cells cultured with antigen or mitogen, but preserved or increased production of IFN-gamma relative to untreated mice. IL-4 and IFN-gamma mRNA associated with CD4+ T lymphocytes isolated from infected lymph nodes showed similar reciprocal changes in response to rMuIL-12 therapy. A single injection of anti-IFN-gamma monoclonal antibody abrogated the protective effect of rMuIL-12 therapy and restored Th2 cytokine responses. We conclude that rMuIL-12 prevents deleterious Th2 T cell responses and promotes curative Th1 responses in an IFN-gamma- dependent fashion during murine leishmaniasis. Since BALB/c leishmaniasis cannot be cured with rMuIFN-gamma alone, additional direct effects of IL-12 during T cell subset selection are suggested. Because rMuIL-12 is uniquely protective in this well-characterized model of chronic parasitism, differences in IL-12 production may underlie heterogenous host responses to L. major and other intracellular pathogens.     

Dendritic cells genetically engineered to express IL-4 exhibit enhanced IL-12p70 production in response to CD40 ligation and accelerate organ allograft rejection

C57BL/10 (B10; H2(b)) bone marrow-derived myeloid dendritic cells (DC) propagated in GM-CSF + IL-4 were transduced with r adenoviral (Ad) vectors encoding either control neomycin-resistance gene (Ad-Neo) or murine IL-4 (Ad-IL-4) on day 5 of culture following CD11c immunomagnetic bead purification. Both Ad-Neo- and Ad-IL-4-transduced DC displayed upregulated surface MHC class II and costimulatory molecules (CD40, CD80, CD86). Ad-IL-4 DC secreted higher levels of bioactive IL-12p70 after CD40 ligation or LPS stimulation than either Ad-Neo or unmodified DC. Only Ad-IL-4 DC produced IL-12p70 in primary MLR, in which they induced augmented proliferative responses of na?ve allogeneic C3H/HeJ (C3H; H2(k)) T-cells. Compared with Ad-Neo DC, Ad-IL-4 DC were also more effective in priming na?ve allogeneic recipients to exhibit specifically enhanced anti-donor T-cell proliferative and CTL responses. T-cells primed in vivo 7 days previously with Ad-IL-4 DC displayed enhanced secretion of Th2 (IL-4, IL-10) but also higher Th1 cytokine (IFNgamma) production following ex vivo challenge with donor alloAg. Moreover, pretreatment of vascularized heart graft recipients with i.v. Ad-IL-4 DC, 1 week before transplant, significantly accelerated rejection and antagonized the therapeutic effect of anti-CD40L (CD154) mAb. These data contrast markedly with recently reported inhibitory effects of autologous Ad-IL-4 DC on autoimmune inflammatory disease.