Intestinal worm infections characteristically induce T-helper 2 cell (Th2) cytokine production. We reviewed studies performed with mice infected with either of two intestinal nematode parasites, Nippostrongylus brasiliensis or Trichinella spiralis, that evaluate the importance of the Th2 cytokine interleukin-4 (IL-4) and IL-13 in protection against these parasites. These studies demonstrate that while IL-4/IL-13 protect against both parasites by activating signal transducer and activator of transcription 6 (Stat6) through IL-4 receptor alpha (IL-4Ralpha) ligation, Stat6 activation protects against these parasites through different mechanisms. Stat6-dependent gene transcription promotes expulsion of N. brasiliensis solely through effects on non-bone marrow-derived cells that may include enhancement of intestinal smooth muscle contractility, changes in intestinal epithelial cell function, and increased intestinal mucus secretion. In contrast, Stat6 signaling promotes immunity to T. spiralis both through effects on bone marrow-derived cells that can be reproduced by treating mice with IL-4 or IL-13 and through effects on non-bone marrow-derived cells. The former effects appear to include T-cell-dependent induction of intestinal mastocytosis, while the latter sensitize non-bone marrow-derived cells to mast cell-produced mediators. We argue that a limited ability of the host immune system to distinguish among different nematode parasites has led to the evolution of a stereotyped Th2 response that activates a set of effector mechanisms that protects against most intestinal nematode parasites.

Studies in mice infected with the gastrointestinal nematode parasite Nippostrongylus brasiliensis demonstrated that IL-4/IL-13 activation of Stat6 suppresses development of intestinal mastocytosis and does not contribute to IL-4/IL-13 production, but is still essential for parasite expulsion. Because expulsion of another gastrointestinal nematode, Trichinella spiralis, unlike N. brasiliensis expulsion, is mast cell dependent, these observations suggested that T. spiralis expulsion would be Stat6 independent. Instead, we find that Stat6 activation by IL-4/IL-13 is required in T. spiralis-infected mice for the mast cell responses that induce worm expulsion and for the cytokine responses that induce intestinal mastocytosis. Furthermore, although IL-4 induces N. brasiliensis expulsion in the absence of B cells, T cells, and mast cells, mast cells and T cells are required for IL-4 induction of T. spiralis expulsion. Thus, Stat6 signaling is required for host protection against N. brasiliensis and T. spiralis but contributes to expulsion of these two worms by different mechanisms. The induction of multiple effector mechanisms by Stat6 signaling provides a way for a cytokine response induced by most gastrointestinal nematode parasites to protect against most of these parasites, even though different effector mechanisms are required for protection against different nematodes.

Resistance to many intestinal nematodes is dependent on the induction of polarized type 2 cytokine responses, whereas type 1 responses can exacerbate these infections. The contributions of IL-4 and IL-13 to the development of resistance have been well described for a variety of intestinal parasites; however, the role of IL-10 has not been previously investigated. In this study we infected IL-10-, IL-10/IL-4-, IL-10/IL-12-, IL-4-, and IL-12-deficient mice with Trichuris muris to determine whether IL-10 contributes to the development of immunity. Interestingly, T. muris-infected IL-10-, IL-4-, and IL-10/IL-4-deficient mice failed to expel the parasite, and animals deficient in IL-10 displayed marked morbidity and mortality. In contrast, double IL-10/IL-12-deficient mice were completely resistant and mounted a highly polarized type 2 cytokine response, demonstrating that the increased susceptibility of IL-10-deficient mice was dependent on IL-12. Further study suggested that the susceptibility of IL-10- and IL-10/IL-4-deficient mice was probably attributable to a marked increase in type 1 cytokine production in those animals. The mortality observed in T. muris-infected IL-10- and IL-10/IL-4-deficient mice correlated with increased inflammation, loss of Paneth cells, and absence of mucus in the cecum. Interestingly, survival was enhanced in T. muris-infected IL-10/IL-4-deficient mice if a broad spectrum antibiotic was administered, suggesting that an outgrowth of opportunistic bacteria was contributing to the high degree of morbidity and mortality. Overall, these studies reveal a critical role for IL-10 in the polarization of Th2 responses, development of resistance during T. muris infection, and maintenance of barrier function in the colon.

Although in vitro development of a Th2 response from naive CD4+ T cells is Stat6 dependent, mice immunized with a goat Ab to mouse IgD have been reported to produce a normal primary IL-4 response in Stat6-deficient mice. Experiments have now been performed with mice immunized with more conventional Ags or inoculated with nematode parasites to account for this apparent discrepancy. The ability of an immunogen to induce a primary in vivo IL-4 response in Stat6-deficient mice was found to vary directly with its ability to induce a strong type 2 cytokine-biased response in normal mice. Even immunogens, however, that induce strong primary IL-4 responses in Stat6-deficient mice induce poor memory IL-4 responses in these mice. Consistent with this, Stat6-deficient CD4+ T cells make relatively normal IL-4 responses when stimulated in vitro for 3 days with anti-CD3 and anti-CD28, but poor IL-4 responses if they are later restimulated with anti-CD3. Thus, Stat6 signaling enhances primary IL-4 responses that are made as part of a type 0 cytokine response (mixed type 1 and type 2) and is required for normal development or survival of Th2 memory cells.

IB kinase (IKK) ␤ is essential for inflammatory cytokine-induced activation of nuclear factor B (NF-B). NF-B plays a pivotal role in the function of major cell types that contribute to the pathophysiological process of rheumatoid arthritis (RA). Here, we report the mechanism and the effect of the IKK␤ inhibitor N- (6-chloro-7-methoxy-9H-␤-carbolin-8-yl)-2-methylnicotinamide (ML120B), a ␤-carboline derivative, on NF-B signaling and gene activation in RA-relevant cell systems. ML120B is a potent, selective, reversible, and ATP-competitive inhibitor of IKK␤ with an IC 50 of 60 nM when evaluated in an IB␣ kinase complex assay. ML120B does not inhibit other IKK isoforms or a panel of other kinases. ML120B concentrationdependently inhibits tumor necrosis factor ␣ (TNF␣)-stimulated NF-B signaling via inhibition of IB␣ phosphorylation, degradation, and NF-B translocation into the nucleus. For the first time, we have demonstrated that in human fibroblast-like synoviocytes, TNF␣-or interleukin (IL)-1␤-induced monocyte chemoattractant protein-1 regulated on activation, normal T cell expressed and secreted and production is IKK␤-dependent. In addition, for the first time, we have demonstrated that lipopolysaccharide-or peptidoglycan-induced cytokine production in human cord blood-derived mast cells is IKK␤-dependent. In addition, in human chondrocytes, ML120B inhibited IL-1␤-induced matrix metalloproteinase production with an IC 50 of approximately 1 M. ML120B also blocked IL-1␤-induced prostaglandin E 2 production. In summary, ML120B blocked numerous NF-B-regulated cell responses that are involved in inflammation and destructive processes in the RA joint. Our findings support the evaluation of IKK␤ inhibitors as anti-inflammatory agents for the treatment of RA.

Expulsion of two gastrointestinal nematode parasites, Nippostrongylus brasiliensis and Trichinella spiralis, is similar in that both require IL-4Rα expression, but different in that T cells and mast cells are required for IL-4-induced expulsion of T. spiralis but not N. brasiliensis. To examine the role of IL-4Rα signaling in immunity to these parasites, we studied worm expulsion in chimeric mice that selectively expressed IL-4Rα on bone marrow-derived or non-bone marrow-derived cells. N. brasiliensis was expelled by mice that expressed IL-4Rα only on non-bone marrow-derived cells, but not by mice that expressed IL-4Rα only on bone marrow-derived cells. Although T. spiralis expulsion required IL-4Rα expression by both bone marrow- and non-bone marrow-derived cells, IL-4 stimulation eliminated the requirement for IL-4Rα expression by bone marrow-derived cells. Thus, direct IL-4Rα signaling of nonimmune gastrointestinal cells may be generally required to induce worm expulsion, even when mast cell and T cell responses are also required.

These preclinical data support using MPP technology to engineer topical formulations to deliver therapeutic drug levels to the back of the eye and could provide major advancements in managing sight-threatening diseases.

IntroductionTopical ophthalmic formulations of corticosteroids are commonly used to treat a variety of ocular diseases and conditions that have an inflammatory component. The purpose of this study was to evaluate the effect of the mucus-penetrating particle (MPP) technology on the pharmacokinetic profile of loteprednol etabonate in the ocular tissues of rabbits.MethodsForty-eight New Zealand White rabbits were randomly assigned to two groups (n = 3 rabbits or 6 eyes per time point) and treated with either the novel loteprednol etabonate MPP suspension formulation, 0.4% (LE-MPP 0.4%), or the commercial Lotemax®-brand loteprednol etabonate ophthalmic suspension, 0.5% (Lotemax 0.5%) (Bausch & Lomb Incorporated, Inc., Rochester, NY, USA). Samples of aqueous humor, various ocular tissues, and plasma were collected from animals over a 12-h period after a single dose of the test articles. Loteprednol etabonate concentrations were assayed using liquid chromatography–tandem mass spectrometry (LC/MS/MS).ResultsLoteprednol etabonate was rapidly absorbed into ocular tissues following administration of either formulation. A higher ocular exposure was achieved using LE-MPP 0.4%, with peak concentrations of approximately threefold higher in ocular tissues and the aqueous humor than Lotemax 0.5%.ConclusionsAdministration of LE-MPP 0.4% improved loteprednol etabonate pharmacokinetic profile in ocular tissues of rabbits. The results of this study support the premise that the MPP technology can be used to enhance ocular exposure for topically applied therapeutic agents. Further studies to assess the clinical efficacy and safety of the LE-MPP formulation are warranted.Electronic supplementary materialThe online version of this article (doi:10.1007/s40123-014-0021-z) contains supplementary material, which is available to authorized users.