TLR-induced cytokines promote effective proinflammatory natural Th17 cell responses.

Naive CD4 lymphocytes undergo a polarization process in the periphery to become induced Th17 (iTh17) cells. Using retinoic acid-related orphan receptor γt (RORγt)-gfp mice, we found that RORγt and the transcription factor promyelocytic leukemia zinc finger (PLZF) are valuable new markers to identify the recently described natural Th17 (nTh17) cell population. nTh17 cells are thymically committed to promptly produce large amounts of IL-17 and IL-22. In this study, we show that, in addition to responding to TCR cross-linking, nTh17 cells secrete IL-17 and IL-22 when stimulated with IL-23 plus IL-1ß, either in recombinant form or in supernatants from TLR4-activated dendritic cells. This innate-like ability of RORγt(+) nTh17 cells to respond to TLR4-induced cytokines was not shared by iTh17 cells. The other distinct properties of RORγt(+) nTh17 cells are their high expression of PLZF and their absence from lamina propria; iTh17 cells are found therein. RORγt(+) nTh17 cells are present in the thymus of germ-free RORγt-gfp and IL-6(-/-) RORΓ: t-gfp mice, indicating that these cells do not require symbiotic microbiota or IL-6 for their generation. Finally, we found that PLZF(+)RORγt(+) nTh17 cells represent one of the primary IL-17-producing innate-like T cell populations in a TLR7 imiquimod model of psoriasis-like disorder, indicating their involvement in this kind of lesion. Collectively, our results reveal RORγt and PLZF as characteristic markers for identifying nTh17 cells and demonstrate one of their novel properties: the ability to respond promptly to TLR-dependent proinflammatory stimuli without TCR engagement, placing them as members of the innate-like T cell family.

Rapamycin combined with TGF-ß converts human invariant NKT cells into suppressive Foxp3+ regulatory cells.

Invariant NKT (iNKT) cells constitute a versatile T cell subset with important regulatory functions, which are thought to result essentially from their capacity to promptly produce cytokines that influence the Th1/Th2 balance. In this study, we report that these cells can also express Foxp3, an important transcriptional regulator associated with suppressive activity, once they have been exposed to TGF-ß. Foxp3 was expressed by iNKT cells from both peripheral and cord blood. CD4(+) iNKT cells acquired Foxp3 expression preferentially, although a lower proportion of their CD4(-) counterpart also became positive. All Foxp3(+) iNKT cells displayed CD25 but not necessarily CTLA4 or GITR, regardless of the upregulation of these markers in the presence of TGF-ß. Exposure to TGF-ß decreased IL-4 and IFN-γ production while increasing IL-10, independently from Foxp3 expression. IL-17 was not detected. TGF-ß induced high levels of Foxp3, but no suppressor activity, which emerged only in the presence of rapamycin. Peripheral and cord blood Foxp3(+) iNKT cells suppressed the proliferation of conventional autologous and heterologous CD4(+) T cells equally, in a cell contact-dependent and Ag-independent manner. Our findings demonstrate that human iNKT cells become suppressive in the presence of TGF-ß plus rapamycin, thus adding a new facet to their complex functional properties.

Identification of an IL-17-producing NK1.1(neg) iNKT cell population involved in airway neutrophilia.

Invariant natural killer T (iNKT) cells are an important source of both T helper type 1 (Th1) and Th2 cytokines, through which they can exert beneficial, as well as deleterious, effects in a variety of inflammatory diseases. This functional heterogeneity raises the question of how far phenotypically distinct subpopulations are responsible for such contrasting activities. In this study, we identify a particular set of iNKT cells that lack the NK1.1 marker (NK1.1(neg)) and secrete high amounts of interleukin (IL)-17 and low levels of interferon (IFN)-gamma and IL-4. NK1.1(neg) iNKT cells produce IL-17 upon synthetic (alpha-galactosylceramide [alpha-GalCer] or PBS-57), as well as natural (lipopolysaccharides or glycolipids derived from Sphingomonas wittichii and Borrelia burgdorferi), ligand stimulation. NK1.1(neg) iNKT cells are more frequent in the lung, which is consistent with a role in the natural immunity to inhaled antigens. Indeed, airway neutrophilia induced by alpha-GalCer or lipopolysaccharide instillation was significantly reduced in iNKT-cell-deficient Jalpha18(-/-) mice, which produced significantly less IL-17 in their bronchoalveolar lavage fluid than wild-type controls. Furthermore, airway neutrophilia was abolished by a single treatment with neutralizing monoclonal antibody against IL-17 before alpha-GalCer administration. Collectively, our findings reveal that NK1.1(neg) iNKT lymphocytes represent a new population of IL-17-producing cells that can contribute to neutrophil recruitment through preferential IL-17 secretion.

Proinflammatory environment dictates the IL-17-producing capacity of human invariant NKT cells.

CD1d-reactive invariant NKT (iNKT) cells have been implicated in a number of experimental models of human pathologies. Given the scope of their immunoregulatory activities mediated through distinct cytokine patterns, it has been proposed that this functional diversity originates from distinct iNKT subpopulations. In this study, we report that human CD161(+) iNKT cells are intrinsically endowed with the capacity to generate IL-17, but require TGF-ß, IL-1ß, and IL-23 to carry out this potential. IL-17-producing iNKT cells are already present in cord blood but, in contrast to peripheral blood iNKT cells, they cannot generate IFN-γ. These IL-17 producers respond to aryl hydrocarbon receptor stimulation and express IL-23 receptor and retinoic acid-related orphan receptor C, similar to conventional T helper 17 cells, from which they differ by their restricted ability to coproduce IL-22. In conclusion, IL-17 production by human iNKT cells depends on two critical parameters, namely an intrinsic program and a proinflammatory environment.

NKT cells are required to induce high IL-33 expression in hepatocytes during ConA-induced acute hepatitis.

Interleukin-33 (IL-33) is thought to be released during cellular death as an alarming cytokine during the acute phase of disease, but its regulation in vivo is poorly understood. We investigated the expression of IL-33 in two mouse models of acute hepatitis by administering either carbon tetrachloride (CCl(4) ) or concanavalin A (ConA). IL-33 was overexpressed in both models but with a stronger induction in ConA-induced hepatitis. IL-33 was weakly expressed in vascular and sinusoidal endothelial cells from normal liver and was clearly induced in CCl(4) -treated mice. Surprisingly, we found that hepatocytes strongly expressed IL-33 exclusively in the ConA model. CD1d knock-out mice, which are deficient in NKT cells and resistant to ConA-induced hepatitis, no longer expressed IL-33 in hepatocytes following ConA administration. Interestingly, invariant NKT (iNKT) cells adoptively transferred into ConA-treated CD1d KO mouse restored IL-33 expression in hepatocytes. This strongly suggests that NKT cells are responsible for the induction of IL-33 in hepatocytes.

Vitronectin dictates intraglomerular fibrinolysis in immune-mediated glomerulonephritis.

During human glomerulonephritis, the severity of injuries correlates with glomerular fibrin deposits, which are tightly regulated by the intraglomerular fibrinolytic system. Here, we evaluated the role of vitronectin (VTN; also known as complement S protein), the principal cofactor of the plasminogen activator inhibitor-1 (PAI-1), in a mouse model of acute glomerulonephritis. We found that in mice subjected to nephrotoxic serum, the absence of VTN resulted in a lower glomerular PAI-1 activity and a higher glomerular fibrinolytic activity. Challenged VTN(-/-) mice displayed significantly less fibrin deposits, proteinuria, and renal failure than their wild-type counterparts. Notably, this protective effect afforded by VTN deficiency was still observed after a C3 depletion. Finally, the injection of VTN(+/+) serum in VTN(-/-) mice induced the glomerular deposition of VTN, increased PAI-1 deposition, decreased glomerular fibrinolytic activity, and aggravated glomerular injury. As in mice, abundant glomerular VTN deposits were also observed in patients with severe glomerulonephritis. Here, we show that plasma-exchange therapy, admittedly beneficial in this clinical context, induces a significant depletion in circulating VTN, which might modulate PAI-1 activity locally and accelerate the clearance of fibrin deposits in the glomeruli. Collectively, these results demonstrate that VTN exerts a deleterious role independently from complement, by directing PAI-dependent fibrinolysis in the glomerular compartment.

AIDS progression is associated with the emergence of IL-17-producing cells early after simian immunodeficiency virus infection.

IL-17 is a potent effector cytokine involved in inflammatory response and antimicrobial defense. We report that SIV infection of rhesus macaques (RMs) results in the emergence of IL-17-expressing cells during the acute phase. This subpopulation appears at day 14 postinfection concomitantly with an increase in TGF-beta and IL-18 expression. This subset, which exhibits phenotypic markers of NK T cells (NKT), rather than Th17 CD4 cells, persists during the chronic phase and is higher in noncontrollers SIV-infected RMs compared with controllers SIV-infected RMs. In contrast, in the nonpathogenic model of SIVagm infection of African green monkeys, no change in the level of IL-17-expressing cells is observed in lymphoid organs. Consistent with the emergence of TGF-beta and IL-18 during the acute phase in SIV-infected RMs, but not in SIV-infected African green monkeys, we demonstrate that in vitro TGF-beta and IL-18 induce the differentiation and expansion of IL-17+NKT+. Altogether, these results demonstrate that IL-17-producing NKT are associated with the pathogenesis of SIV in RMs and suggest that TGF-beta and IL-18 play a role in their development.

Cutting edge: histamine receptor H4 activation positively regulates in vivo IL-4 and IFN-gamma production by invariant NKT cells.

Histamine (HA) is a biogenic amine with multiple activities in the immune system. In this study we demonstrate that histamine-free histidine decarboxylase-deficient (HDC(-/-)) mice present a numerical and functional deficit in invariant NK T (iNKT) cells as evidenced by a drastic decrease of IL-4 and IFN-gamma production. This deficiency was established both by measuring cytokine levels in the serum and intracellularly among gated iNKT cells. It resulted from the lack of HA, because a single injection of this amine into HDC(-/-) mice sufficed to restore normal IL-4 and IFN-gamma production. HA-induced functional recovery was mediated mainly through the H4 histamine receptor (H4R), as assessed by its abrogation after a single injection of a selective H4R antagonist and the demonstration of a similar iNKT cell deficit in H4R(-/-) mice. Our findings identify a novel function of HA through its H4R and suggest that it might become instrumental in modulating iNKT cell functions.

Critical role of ROR-γt in a new thymic pathway leading to IL-17-producing invariant NKT cell differentiation.

Invariant natural killer T (iNKT) cells constitute a subpopulation of T cells that recognize glycolipids presented by CD1d molecules. They are characterized by their prompt production of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma), which enables them to modulate diverse immune responses. Recently, we enlarged this concept by identifying a distinct IL-17-producing iNKT cell subset, named iNKT17 cells. The mechanisms leading to the acquisition of this new iNKT cell activity are unknown. Herein we show that IL-17-producing iNKT cells are already present in the thymus, predominantly among a subset regarded so far as an immature stage of thymic iNKT cell development, the CD1d tetramer(pos)CD44(pos)NK1.1(neg)CD4(neg) cells. Using EGFP reporter mice, we demonstrate that the transcription factor ROR-gammat is critical for the thymic differentiation of this subset because only ROR-gammat(pos) iNKT cells are capable of massively secreting IL-17. Moreover, IL-17-producing CD1d tetramer(pos)CD44(pos)NK1.1(neg)CD4(neg) thymic iNKT cells have reached a mature differentiation stage because they fail to generate other cell subsets in fetal thymic organ culture. Conversely, thymic ROR-gammat(neg) iNKT cell precursors give rise to progeny, but acquire neither ROR-gammat expression nor the ability to secrete IL-17. In conclusion, our findings demonstrate an alternative thymic pathway leading to the development of iNKT17 cells that requires ROR-gammat expression.

Analysis of invariant natural killer T cells in human paracoccidioidomycosis.

Invariant natural killer T (iNKT) cells are capable of recognizing lipid antigens and secreting Th1/Th2 cytokines. Deficiency in iNKT cell number or function has been partially implicated in susceptibility to some infectious diseases, such as tuberculosis. We evaluated iNKT cells in paracoccidioidomycosis, another chronic granulomatous disease endemic in Latin America. iNKT cells were detected using PBS57-loaded tetramer staining and flow cytometry. Circulating iNKT cell numbers were similar among healthy individuals who had previously been cured of paracoccidioidomycosis (susceptible individuals, n = 7) and healthy Paracoccidioides brasiliensis-infected (n = 5) and non-infected individuals (n = 5). iNKT from all three groups expanded similarly upon α-GalCer and a synthetic analog (OCH) stimulation. IFN-γ was the dominant cytokine produced both by ex vivo and by expanded iNKT cells, followed by IL-4 and IL-10, in the three groups. No deficit in the monocyte expression of CD1d was detected. In conclusion, individuals who had developed paracoccidioidomycosis in the past have no impairment in iNKT number, expansion capacity, and cytokine secretion.

Invariant natural killer T cells and TGF-beta attenuate anti-GBM glomerulonephritis.

Invariant natural killer T (iNKT) cells represent a particular subset of T lymphocytes capable of producing several cytokines, which exert regulatory or effector functions, following stimulation of the T cell receptor. In this study, we investigated the influence of iNKT cells on the development of experimental anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). After injection of anti-GBM serum, the number of kidney iNKT cells rapidly increased. iNKT cell-deficient mice (Jalpha18-/-) injected with anti-GBM serum demonstrated worse renal function, increased proteinuria, and greater glomerular and tubular injury compared with similarly treated wild-type mice. We did not detect significant differences in Th1/Th2 polarization in renal tissue that might have explained the severity of disease in Jalpha18-/- mice. Interestingly, expression of both TGF-beta and TGF-beta-induced (TGFBI) mRNA was higher in wild-type kidneys compared with Jalpha18-/- kidneys, suggesting a possible protective role for TGF-beta in anti-GBM GN. Administration of an anti-TGF-beta neutralizing antibody significantly enhanced the severity of disease in wild-type, but not Jalpha18-/-, mice. In conclusion, in experimental anti-GBM GN, iNKT cells attenuate disease severity and TGF-beta has a renoprotective role.

Interleukin-4-dependent innate collaboration between iNKT cells and B-1 B cells controls adaptative contact sensitivity.

We showed that hepatic Valpha14+ invariant natural killer T (iNKT) cells, via their rapid interleukin (IL)-4 production, activate B-1 cells to initiate contact sensitivity (CS). This innate collaboration was absent in IL-4(-/-) and signal transducer and activator of transcription (STAT)-6(-/-) mice and was inhibited by anti-IL-4 treatment. These mice have defective CS because they fail to locally recruit the sensitized effector T cells of acquired immunity. Their CS is reconstituted by transfer of downstream-acting 1-day immune B-1 cells from wild-type mice. Responses were not reconstituted with B-1 cells from IL-4 receptor-alpha(-/-) or STAT-6(-/-) mice, nor by IL-4 treatment of B cell-deficient mice at immunization. Finally, IL-4 was preferentially and transiently produced by hepatic iNKT cells within 7 min after sensitization to mediate collaboration between innate-like iNKT cells and the B-1 B cells that participate in the recruitment of effector T cells in vivo.

TLR-dependent IL-4 production by invariant Valpha14+Jalpha18+ NKT cells to initiate contact sensitivity in vivo.

LPS stimulated B-1 cell polyclonal in vivo IgM responses depend on IL-4 release by invariant Valpha14+Jalpha18+ NKT (iNKT) cells. The IgM Abs can recruit effector T cells to mediate contact sensitivity. LPS activates the B-1 cell response just 1 day later, and depends on CD1d, iNKT cells, IL-4, TLR4, and MyD88. LPS in vivo and in vitro stimulates rapid preferential production of IL-4 in hepatic iNKT cells within 2 h. TLR4 were demonstrated in iNKT cells by flow cytometry and functional studies. Thus, innate microbial stimulation via TLR can activate iNKT cell and B-1 cell collaboration. The result is polyclonal IgM Ab responses capable of recruiting Ag-specific T cells into tissues. This may be involved in the promotion of autoimmunity by infectious agents.

Invariant V alpha 14+ NKT cells participate in the early response to enteric Listeria monocytogenes infection.

Invariant Valpha14(+) NKT cells are a specialized CD1-reactive T cell subset implicated in innate and adaptive immunity. We assessed whether Valpha14(+) NKT cells participated in the immune response against enteric Listeria monocytogenes infection in vivo. Using CD1d tetramers loaded with the synthetic lipid alpha-galactosylceramide (CD1d/alphaGC), we found that splenic and hepatic Valpha14(+) NKT cells in C57BL/6 mice were early producers of IFN-gamma (but not IL-4) after L. monocytogenes infection. Adoptive transfer of Valpha14(+) NKT cells derived from TCRalpha degrees Valpha14-Jalpha18 transgenic (TCRalpha degrees Valpha14Tg) mice into alymphoid Rag(null) gamma(c)(null) mice demonstrated that Valpha14(+) NKT cells were capable of providing early protection against enteric L. monocytogenes infection with systemic production of IFN-gamma and reduction of the bacterial burden in the liver and spleen. Rechallenge experiments demonstrated that previously immunized wild-type and Jalpha18null mice, but not TCRalpha(null) or TCRalpha(null) Valpha14Tg mice, were able to mount adaptive responses to L. monocytogenes. These data demonstrate that Valpha14(+) NKT cells are able to participate in the early response against enteric L. monocytogenes through amplification of IFN-gamma production, but are not essential for, nor capable of, mediating memory responses required to sterilize the host.

Alpha-galactosylceramide-induced iNKT cells suppress experimental allergic asthma in sensitized mice: role of IFN-gamma.

Allergic asthma is a multifaceted syndrome consisting of eosinophil-rich airway inflammation, bronchospasm, and airway hyper-responsiveness (AHR). Using a mouse model of allergic asthma, we previously reported that invariant NKT (iNKT) cells increase the severity of this disease. Herein, we demonstrate that a single i.v. injection of alpha-galactosylceramide (alpha-GalCer), 1 h before the first airway allergen challenge of OVA-sensitized mice, abrogates elicitation of AHR, airway eosinophilia, IL-4 and IL-5 production in bronchoalveolar lavage fluid, and specific anti-OVA IgE antibodies. Further, alpha-GalCer administered intranasally also strongly inhibited the major symptoms of asthma in sensitized and challenged mice. Alpha-GalCer treatment induces iNKT cell accumulation in the lungs, and shifts their cytokine profile from pro-asthmatic IL-4 to a protective IFN-gamma production. The role of IFN-gamma from iNKT cells in protection was shown by adoptive transfer of sorted iNKT cells from OVA-sensitized and alpha-GalCer-treated mice which protected immunized recipients from manifesting asthma by an IFN-gamma-dependent pathway. Our findings demonstrate for the first time that alpha-GalCer administered locally inhibits asthma symptoms, even in predisposed asthmatic mice, through an iNKT cell- and IFN-gamma-dependent pathway.