Upregulation of breathing rate during running exercise by central locomotor circuits in mice.

While respiratory adaptation to exercise is compulsory to cope with the increased metabolic demand, the neural signals at stake remain poorly identified. Using neural circuit tracing and activity interference strategies in mice, we uncover here two systems by which the central locomotor network can enable respiratory augmentation in relation to running activity. One originates in the mesencephalic locomotor region (MLR), a conserved locomotor controller. Through direct projections onto the neurons of the preBötzinger complex that generate the inspiratory rhythm, the MLR can trigger a moderate increase of respiratory frequency, prior to, or even in the absence of, locomotion. The other is the lumbar enlargement of the spinal cord containing the hindlimb motor circuits. When activated, and through projections onto the retrotrapezoid nucleus (RTN), it also potently upregulates breathing rate. On top of identifying critical underpinnings for respiratory hyperpnea, these data also expand the functional implication of cell types and pathways that are typically regarded as "locomotor" or "respiratory" related.

A medullary centre for lapping in mice.

It has long been known that orofacial movements for feeding can be triggered, coordinated, and often rhythmically organized at the level of the brainstem, without input from higher centers. We uncover two nuclei that can organize the movements for ingesting fluids in mice. These neuronal groups, IRtPhox2b and Peri5Atoh1, are marked by expression of the pan-autonomic homeobox gene Phox2b and are located, respectively, in the intermediate reticular formation of the medulla and around the motor nucleus of the trigeminal nerve. They are premotor to all jaw-opening and tongue muscles. Stimulation of either, in awake animals, opens the jaw, while IRtPhox2b alone also protracts the tongue. Moreover, stationary stimulation of IRtPhox2b entrains a rhythmic alternation of tongue protraction and retraction, synchronized with jaw opening and closing, that mimics lapping. Finally, fiber photometric recordings show that IRtPhox2b is active during volitional lapping. Our study identifies one of the subcortical nuclei underpinning a stereotyped feeding behavior.

Absent phasing of respiratory and locomotor rhythms in running mice.

Examining whether and how the rhythms of limb and breathing movements interact is highly informative about the mechanistic origin of hyperpnoea during running exercise. However, studies have failed to reveal regularities. In particular, whether breathing frequency is inherently proportional to limb velocity and imposed by a synchronization of breaths to strides is still unclear. Here, we examined respiratory changes during running in the resourceful mouse model. We show that, for a wide range of trotting speeds on a treadmill, respiratory rate increases to a fixed and stable value irrespective of trotting velocities. Respiratory rate was yet further increased during escape-like running and most particularly at gallop. However, we found no temporal coordination of breaths to strides at any speed, intensity, or gait. Our work thus highlights that exercise hyperpnoea can operate, at least in mice and in the presently examined running regimes, without phasic constraints from limb movements.

SLAM-associated protein favors the development of iNKT2 over iNKT17 cells.

Invariant NKT (iNKT) cells differentiate in the thymus into three distinct lineages defined by their cytokine and transcription factor expression. Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) is essential for early stages of iNKT cell development, but its role during terminal differentiation of iNKT1, iNKT2, or iNKT17 cells remains unclear. Taking advantage of SAP-deficient mice expressing a Vα14-Jα18 TCRα transgene, we found that SAP is critical not only for IL-4 production but also for the terminal differentiation of IL-4-producing iNKT2 cells. Furthermore, without SAP, the IL-17 producing subset is expanded, while IFN-γ-producing iNKT1 differentiation is only moderately compromised. Lack of SAP reduced the expression of the transcription factors GATA-3 and promyelocytic leukemia zinc finger, but enhanced the levels of retinoic acid receptor-related orphan receptor γt. In the absence of SAP, lineage commitment was actually shifted toward the emergence of iNKT17 over iNKT2 cells. Collectively, our data unveil a new critical regulatory function for SAP in thymic iNKT cell fate decisions.

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.

IL-17-producing invariant NKT cells in lymphoid organs are recent thymic emigrants identified by neuropilin-1 expression.

Despite increasing knowledge on the mechanisms of invariant natural killer T (iNKT)-cell development in the thymus, the function of recent thymic emigrant (RTE) iNKT cells remains largely unexplored, principally because of a lack of bona fide markers to distinguish RTE from long-lived iNKT cells. Whether the recently described IL-17-producing iNKT cell subset is part of RTE has notably not been addressed. In the present study, we show that neuropilin-1 (Nrp-1), a transmembrane receptor mainly found on T-regulatory (Treg) cells in the murine immune system, is specifically expressed on RTE iNKT cells in naive mice. We used the Nrp-1 marker to discriminate RTE from mature iNKT cells and compare their functions. We show that RTE iNKT cells proliferate more than mature iNKT cells after in vitro activation; that, unlike mature iNKT cells, most RTE iNKT cells fail to rapidly produce IFN-γ and IL-4 after in vivo activation; and, most importantly, that IL-17-producing iNKT cells in lymphoid organs of naive mice are contained within the RTE iNKT cell pool. Our results establish an accurate marker of RTE iNKT cells and reveal that continuous thymic output is required for pro-inflammatory IL-17 secretion, a key function of adult iNKT cells.

Cutting edge: intravenous Ig inhibits invariant NKT cell-mediated allergic airway inflammation through FcγRIIIA-dependent mechanisms.

Despite their increasing use in autoimmune, inflammatory, and allergic conditions, the mechanism of action of i.v. Igs (IVIg) is poorly understood. On the basis of the critical role of invariant NKT (iNKT) cells in allergic airway inflammation (AAI) and their constitutive expression of the low-affinity IgG receptor FcγRIIIA, we surmised that IVIg targets iNKT cells to exert their anti-inflammatory effect. We found that IVIg treatment significantly inhibited AAI in OVA-sensitized C57BL/6 mice and downregulated α-galactosylceramide-induced iNKT cell activation and cytokine production. Allergic responses were restored in iNKT cell-deficient mice by transferring iNKT cells from PBS- but not from IVIg-treated mice, suggesting that IVIg acts directly on activated iNKT cells that have a critical role in AAI. The inhibitory effects of IVIg on both iNKT cell activation/function and OVA-driven AAI were lost in FcγRIIIA(-/-) mice. Our data unravel an FcγRIIIA-dependent inhibitory effect of IVIg on activated iNKT cells that confers protection in AAI.

A natural protective function of invariant NKT cells in a mouse model of innate-cell-driven lung inflammation.

Activation of invariant natural killer T (iNKT) cells by treatment with their α-galactosyl ceramide ligand provides therapeutic benefits in several immune inflammatory settings. Given the artificial nature of this stimulation, the natural regulatory functions of iNKT remain uncertain. Addressing this issue in a mouse model of innate-cell-driven lung inflammation induced by the cytokine/alarmin IL-33 that targets iNKT cells, we found that eosinophil and neutrophil recruitment was markedly increased in treated iNKT cell-deficient (Jα18 KO) mice, as was the local production of eotaxin and keratinocyte chemoattractant chemokines. By contrast, lung inflammation decreased after adoptive transfer of iNKT cells, which restored the WT inflammatory response in Jα18 KO mice. Finally, we established that this natural anti-inflammatory function of iNKT cells depends on their IFN-γ production and on endogenous IL-12. Our study provides the first evidence of a protective role of iNKT cells during lung inflammation that does not require pharmacological TCR engagement.

The pro-Th2 cytokine IL-33 directly interacts with invariant NKT and NK cells to induce IFN-gamma production.

IL-33 has recently been identified as a cytokine endowed with pro-Th2 functions, raising the question of its effect on invariant natural killer T cell (iNKT), which are potent IL-4 producers. Here, we report a two-fold increase of iNKT-cell counts in spleen and liver after a 7-day treatment of mice with IL-33, which results from a direct effect, given that purified iNKT cells express the T1/ST2 receptor constitutively and respond to IL-33 by in vitro expansion and functional activation. Conversely to the expected pro-Th2 effect, IL-33 induced a preferential increase in IFN-gamma rather than IL-4 production upon TCR engagement that depended on endogenous IL-12. Moreover, in combination with the pro-inflammatory cytokine IL-12, IL-33 enhanced IFN-gamma production by both iNKT and NK cells. Taken together these data support the conclusion that IL-33 can contribute as a co-stimulatory factor to innate cellular immune responses.

Invariant NKT cells inhibit development of the Th17 lineage.

T cells differentiate into functionally distinct effector subsets in response to pathogen encounter. Cells of the innate immune system direct this process; CD1d-restricted invariant natural killer T (iNKT) cells, for example, can either promote or inhibit Th(1) and Th(2) responses. Recently, a new subset of CD4(+) T helper cells, called Th(17), was identified that is implicated in mucosal immunity and autoimmune disorders. To investigate the influence of iNKT cells on the differentiation of naïve T cells we used an adoptive transfer model of traceable antigen-specific CD4(+) T cells. Transferred naïve CD25(-)CD62L(+) CD4(+) T cells were primed by antigen immunization of the recipient mice, permitting their expansion and Th(17) differentiation. This study establishes that in vivo activation of iNKT cells during T-cell priming impedes the commitment of naïve T cells to the Th(17) lineage. In vivo cytokine neutralization experiments revealed a role for IL-4, IL-10, and IFN-gamma in the iNKT-cell-mediated regulation of T-cell lineage development. Moreover, by comparing IL-17 production by antigen-experienced T cells from unmanipulated wild-type mice and iNKT-cell-deficient mice, we demonstrate an enhanced Th(17) response in mice lacking iNKT cells. This invigorated Th(17) response reverts to physiological levels when iNKT cells are introduced into Jalpha18(-/-) mice by adoptive transfer, indicating that iNKT cells control the Th(17) compartment at steady state. We conclude that iNKT cells play an important role in limiting development of the Th(17) lineage and suggest that iNKT cells provide a natural barrier against Th(17) responses.

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.

Ginger prevents Th2-mediated immune responses in a mouse model of airway inflammation.

It is well documented that compounds from rhizomes of Zingiber officinale, commonly called ginger, have anti-inflammatory properties. Here, we show that ginger can exert such functions in vivo, namely in a mouse model of Th2-mediated pulmonary inflammation. The preparation of ginger aqueous extract (Zo.Aq) was characterized by mass spectrometry as an enriched fraction of n-gingerols. Intraperitoneal injections of this extract before airway challenge of ovalbumin (OVA)-sensitized mice resulted in a marked decrease in the recruitment of eosinophils to the lungs as attested by cell counts in bronchoalveolar lavage (BAL) fluids and histological examination. Resolution of airway inflammation induced by Zo.Aq was accompanied by a suppression of the Th2 cell-driven response to allergen in vivo. Thus, IL-4, IL-5 and eotaxin levels in the lungs as well as specific IgE titres in serum were clearly diminished in ginger-treated mice relative to their controls after allergen sensitization and challenge. Finally, we found that [6]-gingerol, a major constituent of ginger, was sufficient to suppress eosinophilia in our model of inflammation. This is the first evidence that ginger can suppress Th2-mediated immune responses and might thus provide a possible therapeutic application in allergic asthma.

The Pro-Th1 cytokine IL-12 enhances IL-4 production by invariant NKT cells: relevance for T cell-mediated hepatitis.

IL-12 is essential for invariant NKT (iNKT) cells because it can maintain a functionally active population and promote a cytokine profile that is assumed to be mainly of the pro-Th1 type. We used the murine concanavalin A (Con A)-induced hepatitis model, in which iNKT cells, IL-12, IL-4, and IFN-gamma are equally requisite, to reevaluate this issue. We demonstrate that IL-12 interacts directly with iNKT cells, contributes to their recruitment to the liver, and enhances their IL-4 production, which is essential for disease onset. IL-12-deficient mice were less susceptible to experimental hepatitis and their iNKT cells produced less IL-4 than their wild-type counterpart. A normal response could be restored by IL-12 injection, revealing its importance as endogenous mediator. In accordance with this observation, we found that iNKT cells expressed the IL-12R constitutively, in contrast to conventional T cells. Furthermore, the physiological relevance of our data is supported by the lower susceptibility to disease induction of NOD mice, known for their inherent functional and numerical abnormalities of iNKT cells associated with decreased iNKT cell-derived IL-4 production and low IL-12 secretion. Taken together, our findings provide the first evidence that IL-12 can enhance the immune response through increased IL-4 production by iNKT cells, underscoring once more the functional plasticity of this subset.

Activation of invariant NK T cells protects against experimental rheumatoid arthritis by an IL-10-dependent pathway.

Invariant natural killer T (iNKT) cells are a unique lymphocyte subtype implicated in the regulation of autoimmunity and a good source of protective Th2 cytokines. Agonist alpha-galactosylceramide (alpha-GalCer) of iNKT cells exert a therapeutical effect in type 1 diabetes. We investigated whether iNKT activation with alpha-GalCer was protective in collagen-induced arthritis (CIA) in DBA/1 mice, a standard model of rheumatoid arthritis. Here, we have shown that in vivo iNKT cell function was altered in DBA/1 mice since stimulation with alpha-GalCer led to decreased IL-4 and IFN-gamma levels in sera, as compared with C57BL/6 mice. alpha-GalCer induced a clear-cut diminution of clinical and histological arthritides. An anti-IL-10 receptor antibody abrogated the protective effect of alpha-GalCer, suggesting a key role for IL-10 in the protection against CIA by activated iNKT cells. Confirming these data, disease protection conferred by alpha-GalCer correlated with the ability of LN CD4+ cells to secrete larger amounts of IL-10. These findings suggest that in CIA susceptibility to autoimmunity is associated with dysfunctions of iNKT cells. Our demonstration that iNKT cell activation by alpha-GalCer remains efficient in CIA-prone DBA/1 mice to provide protective IL-10 suggests that this could be used therapeutically to treat autoimmune arthritis.

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.

Relevance of sexual dimorphism to regulatory T cells: estradiol promotes IFN-gamma production by invariant natural killer T cells.

Mechanisms accounting for gender dimorphism during immune responses are still poorly understood. Since invariant natural killer T (iNKT) cells exert important regulatory functions through their capacity to produce both T helper 1 (Th1) and Th2 cytokines, we addressed the question of whether these activities could be modulated by sexual hormones. We found that in vivo challenge with the specific ligand of iNKT cells, alpha-galactosylceramide (alpha-GalCer), induced significantly higher concentrations of interferon gamma (IFN-gamma) in the serum of female than in that of male mice, while interleukin 4 (IL-4) production was not modified. In support of a crucial role of ovarian hormones in this phenomenon, a significant decrease of serum IFN-gamma concentrations occurred in ovariectomized females, in response to treatment with alpha-GalCer, while orchidectomy affected neither IFN-gamma nor IL-4 serum concentrations in males. The implication of estrogens in this selective enhancement of IFN-gamma production by iNKT cells was demonstrated by (1) the increased alpha-GalCer-induced IFN-gamma synthesis by iNKT cells upon both in vitro and in vivo exposure to estradiol and (2) the abolition of the sex-linked difference in alpha-GalCer-induced IFN-gamma release in estrogen receptor alpha-deficient mice. These results provide the first evidence that estrogens influence iNKT cells leading to this gender dimorphism in their cytokine production profile.

Exacerbated Th2-mediated airway inflammation and hyperresponsiveness in autoimmune diabetes-prone NOD mice: a critical role for CD1d-dependent NKT cells.

The NOD mouse has proved to be a relevant model of insulin-dependent diabetes mellitus, closely resembling the human disease. However, it is unknown whether this strain presents a general biastoward Th1-mediated autoimmunity or remains capable of mounting complete Th2-mediated responses. Here, we show that NOD mice have the capacity to develop a typical Th2-mediated disease, namely experimental allergic asthma. In contrast to what might have been expected, they even developed a stronger Th2-mediated pulmonary inflammatory response than BALB/c mice, a strain that shows a typical Th2 bias in this model. Thus, after allergen sensitization and intra-nasal challenge, the typical features of experimental asthma were exacerbated in NOD mice, including enhanced bronchopulmonary responsiveness, mucus production and eosinophilic inflammation in the lungs as well as specific IgE titers in serum. These hallmarks of allergic asthma were associated with increased IL-4, IL-5, IL-13 and eotaxin production in the lungs, as compared with BALB/c mice. Notwithstanding their quantitative and functional defect in NOD mice, CD1d-dependent NKT cells contribute to aggravate the disease, since in OVA-immunized CD1d(-/-) NOD mice, which are deficient in this particular T cell subset, airway eosinophilia was clearly diminished relative to NOD littermates. This is the first evidence that autoimmune diabetes-prone NOD mice can also give rise to enhanced Th2-mediated responses and might thus provide a useful model for the study of common genetic and cellular components, including NKT cells that contribute to both asthma and type 1 diabetes.

Cutting edge: invariant V alpha 14 NKT cells are required for allergen-induced airway inflammation and hyperreactivity in an experimental asthma model.

Airway hyperreactivity (AHR), eosinophilic inflammation with a Th2-type cytokine profile, and specific Th2-mediated IgE production characterize allergic asthma. In this paper, we show that OVA-immunized Jalpha18(-/-) mice, which are exclusively deficient in the invariant Valpha14(+) (iValpha14), CD1d-restricted NKT cells, exhibit impaired AHR and airway eosinophilia, decreased IL-4 and IL-5 production in bronchoalveolar lavage fluid, and reduced OVA-specific IgE compared with wild-type (WT) littermates. Adoptive transfer of WT iValpha14 NKT cells fully reconstitutes the capacity of Jalpha18(-/-) mice to develop allergic asthma. Also, specific tetramer staining shows that OVA-immunized WT mice have activated (CD69(+)) iValpha14 NKT cells. Importantly, anti-CD1d mAb treatment blocked the ability of iValpha14 T cells to amplify eosinophil recruitment to airways, and both Th2 cytokine and IgE production following OVA challenge. In conclusion, these findings clearly demonstrate that iValpha14 NKT cells are required to participate in allergen-induced Th2 airway inflammation through a CD1d-dependent mechanism.