IgE-activated mast cells enhance TLR4-mediated antigen-specific CD4+ T cell responses.

Mast cells are potent mediators of allergy and asthma, yet their role in regulating adaptive immunity remains ambiguous. On the surface of mast cells, the crosslinking of IgE bound to FcεRI by a specific antigen recognized by that IgE triggers the release of immune mediators such as histamine and cytokines capable of activating other immune cells; however, little is known about the mast cell contribution to the induction of endogenous, antigen-specific CD4+ T cells. Here we examined the effects of specific mast cell activation in vivo on the initiation of an antigen-specific CD4+ T cell response. While CD4+ T cells were not enhanced by FcεRI stimulation alone, their activation was synergistically enhanced when FcεRI activation was combined with TLR4 stimulation. This enhanced activation was dependent on global TLR4 stimulation but appeared to be less dependent on mast cell expressed TLR4. This study provides important new evidence to support the role of mast cells as mediators of the antigen-specific adaptive immune response.

Nontypeable Haemophilus influenzae Type IV Pilus Mediates Augmented Adherence to Rhinovirus-Infected Human Airway Epithelial Cells.

Human rhinovirus (hRV) is frequently detected in the upper respiratory tract, and symptomatic infection is associated with an increased nasopharyngeal bacterial load, with subsequent development of secondary bacterial diseases. Nontypeable Haemophilus influenzae (NTHI) is a commensal bacterial species of the human nasopharynx; however, in the context of prior or concurrent upper respiratory tract viral infection, this bacterium commonly causes multiple diseases throughout the upper and lower respiratory tracts. The present study was conducted to determine the mechanism(s) by which hRV infection promotes the development of NTHI-induced diseases. We showed that hRV infection of polarized primary human airway epithelial cells resulted in increased adherence of NTHI, due in part to augmented expression of CEACAM1 and ICAM1, host cell receptors to which NTHI binds via engagement of multiple adhesins. Antibody blockade of these host cell receptors significantly reduced NTHI adherence. With a specific focus on the NTHI type IV pilus (T4P), which we have previously shown binds to ICAM1, an essential adhesin and virulence determinant, we next showed that T4P-directed antibody blockade significantly reduced NTHI adherence to hRV-infected airway cells and, further, that expression of this adhesin was required for the enhanced adherence observed. Collectively, these data provide a mechanism by which "the common cold" promotes diseases due to NTHI, and they add further support for the use of PilA (the majority subunit of T4P) as a vaccine antigen, since antibodies directed against PilA are expected to limit the notably increased bacterial load associated with hRV coinfection and thereby to prevent secondary NTHI-induced diseases of the respiratory tract.

TIM-3-Expressing Mast Cells Are Present in Chronic Rhinosinusitis with Nasal Polyps.

Objectives To identify whether TIM-3 expression is present in the mast cell population within nasal polyps and to determine its correlation with clinical severity in patients with chronic rhinosinusitis with nasal polyposis. Study Design Basic science, translational study. Setting Nasal polyp tissue collected from patients seen at a tertiary care hospital (2015-2016). Subjects and Methods Nasal polyp tissue obtained during functional endoscopic sinus surgery (n = 24) was enzymatically digested into epithelial and stromal fractions. Viable mast cells expressing TIM-3 were identified using flow cytometry for the following: CD45, Live/Dead, c-kit, FcεR1, TIM-3. Disease severity was assessed using the Sino-Nasal Outcome Test, Lund-Mackay staging system, Lund-Kennedy staging system, and complete blood counts. Results Mast cells were found in both the epithelial and stromal layers of polyps, with a greater %TIM-3+ mast cells in the epithelial layer compared with that of the stromal layer ( P = .001). As the percentage of mast cells increased, there was a comparative worsening in endoscopic severity after comparing pre- and postoperative LK scores (ρ = -0.455, P = .029). In a subgroup of patients with concomitant asthma, increased epithelial %TIM-3+ mast cells also correlated with worsening endoscopic appearance postoperatively (ρ = 0.866, P = .001, n = 11). Oral corticosteroid treatment did not change the viability of mast cells nor their influence on the increased postoperative endoscopic disease severity (ρ = -0.544, P = .020, n = 18). Conclusion Viable mast cells were found to be present in polyps with increased TIM-3 expression at the epithelial layer. This suggests that TIM-3 may play a role in chronic inflammation in CRSwNP via mast cell activation.

Cutting Edge: Murine Mast Cells Rapidly Modulate Metabolic Pathways Essential for Distinct Effector Functions.

There is growing appreciation that cellular metabolic and bioenergetic pathways do not play merely passive roles in activated leukocytes. Rather, metabolism has important roles in controlling cellular activation, differentiation, survival, and effector function. Much of this work has been performed in T cells; however, there is still very little information regarding mast cell metabolic reprogramming and its effect on cellular function. Mast cells perform important barrier functions and help control type 2 immune responses. In this study we show that murine bone marrow-derived mast cells rapidly alter their metabolism in response to stimulation through the FcεRI. We also demonstrate that specific metabolic pathways appear to be differentially required for the control of mast cell function. Manipulation of metabolic pathways may represent a novel point for the manipulation of mast cell activation.

Mast cell activation is enhanced by Tim1:Tim4 interaction but not by Tim-1 antibodies.

Polymorphisms in the T cell (or transmembrane) immunoglobulin and mucin domain 1 ( TIM-1) gene, particularly in the mucin domain, have been associated with atopy and allergic diseases in mice and human. Genetic- and antibody-mediated studies revealed that Tim-1 functions as a positive regulator of Th2 responses, while certain antibodies to Tim-1 can exacerbate or reduce allergic lung inflammation. Tim-1 can also positively regulate the function of B cells, NKT cells, dendritic cells and mast cells. However, the precise molecular mechanisms by which Tim-1 modulates immune cell function are currently unknown. In this study, we have focused on defining Tim-1-mediated signaling pathways that enhance mast cell activation through the high affinity IgE receptor (FceRI). Using a Tim-1 mouse model lacking the mucin domain (Tim-1 Dmucin), we show for the first time that the polymorphic Tim-1 mucin region is dispensable for normal mast cell activation. We further show that Tim-4 cross-linking of Tim-1 enhances select signaling pathways downstream of FceRI in mast cells, including mTOR-dependent signaling, leading to increased cytokine production but without affecting degranulation.

Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation.

T cell (or transmembrane) immunoglobulin and mucin domain protein 3 (Tim-3) has attracted significant attention as a novel immune checkpoint receptor (ICR) on chronically stimulated, often dysfunctional, T cells. Antibodies to Tim-3 can enhance antiviral and antitumor immune responses. Tim-3 is also constitutively expressed by mast cells, NK cells and specific subsets of macrophages and dendritic cells. There is ample evidence for a positive role for Tim-3 in these latter cell types, which is at odds with the model of Tim-3 as an inhibitory molecule on T cells. At this point, little is known about the molecular mechanisms by which Tim-3 regulates the function of T cells or other cell types. We have focused on defining the effects of Tim-3 ligation on mast cell activation, as these cells constitutively express Tim-3 and are activated through an ITAM-containing receptor for IgE (FcεRI), using signaling pathways analogous to those in T cells. Using a variety of gain- and loss-of-function approaches, we find that Tim-3 acts at a receptor-proximal point to enhance Lyn kinase-dependent signaling pathways that modulate both immediate-phase degranulation and late-phase cytokine production downstream of FcεRI ligation.

T cell receptor-dependent activation of mTOR signaling in T cells is mediated by Carma1 and MALT1, but not Bcl10.

Signaling to the mechanistic target of rapamycin (mTOR) regulates diverse cellular processes, including protein translation, cellular proliferation, metabolism, and autophagy. Most models place Akt upstream of the mTOR complex, mTORC1; however, in T cells, Akt may not be necessary for mTORC1 activation. We found that the adaptor protein Carma1 [caspase recruitment domain (CARD)-containing membrane-associated protein 1] and at least one of its associated proteins, the paracaspase MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), were required for optimal activation of mTOR in T cells in response to stimulation of the T cell receptor (TCR) and the co-receptor CD28. However, Bcl10, which binds to Carma1 and MALT1 to form a complex that mediates signals from the TCR to the transcription factor NF-κB (nuclear factor κB), was not required. The catalytic activity of MALT1 was required for the proliferation of stimulated CD4+ T cells, but not for early TCR-dependent activation events. Consistent with an effect on mTOR, MALT1 activity was required for the increased metabolic flux in activated CD4+ T cells. Together, our data suggest that Carma1 and MALT1 play previously unappreciated roles in the activation of mTOR signaling in T cells after engagement of the TCR.

Regulation of fertility, survival, and cuticle collagen function by the Caenorhabditis elegans eaf-1 and ell-1 genes.

EAF2, an androgen-regulated protein, interacts with members of the ELL (eleven-nineteen lysine-rich leukemia) transcription factor family and also acts as a tumor suppressor. Although these proteins control transcriptional elongation and perhaps modulate the effects of other transcription factors, the mechanisms of their actions remain largely unknown. To gain new insights into the biology of the EAF2 and ELL family proteins, we used Caenorhabditis elegans as a model to explore the in vivo roles of their worm orthologs. Through the use of transgenic worms, RNAi, and an eaf-1 mutant, we found that both genes are expressed in multiple cell types throughout the worm life cycle and that they play important roles in fertility, survival, and body size regulation. ELL-1 and EAF-1 likely contribute to these activities in part through modulating cuticle synthesis, given that we observed a disrupted cuticle structure in ell-1 RNAi-treated or eaf-1 mutant worms. Consistent with disruption of cuticle structure, loss of either ELL-1 or EAF-1 suppressed the rol phenotype of specific collagen mutants, possibly through the control of dpy-3, dpy-13, and sqt-3 collagen gene expression. Furthermore, we also noted the regulation of collagen expression by ELL overexpression in PC3 human prostate cancer cells. Together, these results reveal important roles for the eaf-1 and ell-1 genes in the regulation of extracellular matrix components.

Akt fine-tunes NF-κB-dependent gene expression during T cell activation.

Activation of the NF-κB signaling pathway is critical for leukocyte activation and development. Although previous studies suggested a role for the Akt kinase in coupling the T cell antigen receptor and CD28 to NF-κB activation in T cells, the nature of the role of Akt in this pathway is still unclear. Using a targeted gene profiling approach, we found that a subset of NF-κB-dependent genes required Akt for optimal up-regulation during T cell activation. The selective effects of Akt were manifest at the level of mRNA transcription and p65/RelA binding to upstream promoters and appear to be due to altered formation of the Carma1-Bcl10 complex. The proinflammatory cytokine TNF-α was found to be particularly sensitive to Akt inhibition or knockdown, including in primary human blood T cells and a murine model of rheumatoid arthritis. Our findings are consistent with a hierarchy in the expression of NF-κB-dependent genes, controlled by the strength and/or duration of NF-κB signaling. More broadly, our results suggest that defining the more graded effects of signaling, such as those demonstrated here for Akt and the NF-κB pathway, is important to understanding how cells can fine-tune signaling responses for optimal sensitivity and specificity.