Deficiency of UCHL1 results in insufficient decidualization accompanied by impaired dNK modulation and eventually miscarriage.

BACKGROUND: Miscarriage is a frustrating complication of pregnancy that is common among women of reproductive age. Insufficient decidualization which not only impairs embryo implantation but disturbs fetomaternal immune-tolerance, has been widely regarded as a major cause of miscarriage; however, the underlying mechanisms resulting in decidual impairment are largely unknown. METHODS: With informed consent, decidual tissue from patients with spontaneous abortion or normal pregnant women was collected to detect the expression profile of UCHL1. Human endometrial stromal cells (HESCs) were used to explore the roles of UCHL1 in decidualization and dNK modulation, as well as the mechanisms involved. C57/BL6 female mice (7-10 weeks old) were used to construct pregnancy model or artificially induced decidualization model to evaluate the effect of UCHL1 on mice decidualization and pregnancy outcome. RESULTS: The Ubiquitin C-terminal hydrolase L1 (UCHL1), as a deubiquitinating enzyme, was significantly downregulated in decidua from patients with miscarriage, along with impaired decidualization and decreased dNKs. Blockage of UCHL1 led to insufficient decidualization and resultant decreased expression of cytokines CXCL12, IL-15, TGF-ß which were critical for generation of decidual NK cells (dNKs), whereas UCHL1 overexpression enhanced decidualization accompanied by increase in dNKs. Mechanistically, the promotion of UCHL1 on decidualization was dependent on its deubiquitinating activity, and intervention of UCHL1 inhibited the activation of JAK2/STAT3 signaling pathway, resulting in aberrant decidualization and decreased production of cytokines associated with dNKs modulation. Furthermore, we found that inhibition of UCHL1 also disrupted the decidualization in mice and eventually caused adverse pregnancy outcome. CONCLUSIONS: UCHL1 plays significant roles in decidualization and dNKs modulation during pregnancy in both humans and mice. Its deficiency indicates a poor pregnancy outcome due to defective decidualization, making UCHL1 a potential target for the diagnosis and treatment of miscarriage.

Spray-Dried and Spray-Freeze-Dried Powder Formulations of an Anti-Interleukin-4Rα Antibody for Pulmonary Delivery.

OBJECTIVE: The therapeutic options for severe asthma are limited, and the biological therapies are all parenterally administered. The purpose of this study was to formulate a monoclonal antibody that targets the receptor for IL-4, an interleukin implicated in the pathogenesis of severe asthma, into a dry powder intended for delivery via inhalation. METHODS: Dehydration was achieved using either spray drying or spray freeze drying, which exposes the thermolabile biomacromolecules to stresses such as shear and adverse temperatures. 2-hydroxypropyl-beta-cyclodextrin was incorporated into the formulation as protein stabiliser and aerosol performance enhancer. The powder formulations were characterised in terms of physical and aerodynamic properties, while the antibody was assessed with regard to its structural stability, antigen-binding ability, and in vitro biological activity after drying. RESULTS: The spray-freeze-dried formulations exhibited satisfactory aerosol performance, with emitted fraction exceeding 80% and fine particle fraction of around 50%. The aerosolisation of the spray-dried powders was hindered possibly by high residual moisture. Nevertheless, the antigen-binding ability and inhibitory potency were unaffected for the antibody in the selected spray-dried and spray-freeze-dried formulations, and the antibody was physically stable even after one-year storage at ambient conditions. CONCLUSIONS: The findings of this study establish the feasibility of developing an inhaled dry powder formulation of an anti-IL-4R antibody using spray drying and spray freeze drying techniques with potential for the treatment of severe asthma.

PLZF-expressing CD4 T cells show the characteristics of terminally differentiated effector memory CD4 T cells in humans.

We show the presence of lymphoid tissue-resident PLZF+ CD45RA+ RO+ CD4 T cells in humans. They express HLA-DR, granzyme B, and perforin and are low on CCR7 like terminally differentiated effector memory (Temra) cells and are likely generated from effector T cells (Te) or from central (Tcm) or effector (Tem) memory T (Tcm) cells during immune responses. Tn, Naïve T cells.

A transgenic TCR directs the development of IL-4+ and PLZF+ innate CD4 T cells.

MHC class II-expressing thymocytes can efficiently mediate positive selection of CD4 T cells in mice. Thymocyte-selected CD4 (T-CD4) T cells have an innate-like phenotype similar to invariant NKT cells. To investigate the development and function of T-CD4 T cells in-depth, we cloned TCR genes from T-CD4 T cells and generated transgenic mice. Remarkably, positive selection of T-CD4 TCR transgenic (T3) thymocytes occurred more efficiently when MHC class II was expressed by thymocytes than by thymic epithelial cells. Similar to polyclonal T-CD4 T cells and also invariant NKT cells, T3 CD4 T cell development is controlled by signaling lymphocyte activation molecule/signaling lymphocyte activation molecule-associated protein signaling, and the cells expressed both IL-4 and promyelocytic leukemia zinc finger (PLZF). Surprisingly, the selected T3 CD4 T cells were heterogeneous in that only half expressed IL-4 and only half expressed PLZF. IL-4- and PLZF-expressing cells were first found at the double-positive cell stage. Thus, the expression of IL-4 and PLZF seems to be determined by an unidentified event that occurs postselection and is not solely dependent on TCR specificity or the selection process, per se. Taken together, our data show for the first time, to our knowledge, that the TCR specificity regulates but does not determine the development of innate CD4 T cells by thymocytes.

Development of promyelocytic leukemia zinc finger-expressing innate CD4 T cells requires stronger T-cell receptor signals than conventional CD4 T cells.

MHC class II-expressing thymocytes and thymic epithelial cells can mediate CD4 T-cell selection resulting in functionally distinct thymocyte-selected CD4 (T-CD4) and epithelial-selected CD4 (E-CD4) T cells, respectively. However, little is known about how T-cell receptor (TCR) signaling influences the development of these two CD4 T-cell subsets. To study TCR signaling for T-CD4 T-cell development, we used a GFP reporter system of Nur77 in which GFP intensity directly correlates with TCR signaling strength. T-CD4 T cells expressed higher levels of GFP than E-CD4 T cells, suggesting that T-CD4 T cells received stronger TCR signaling than E-CD4 T cells during selection. Elimination of Ras GTPase-activating protein enhanced E-CD4 but decreased T-CD4 T-cell selection efficiency, suggesting a shift to negative selection. Conversely, the absence of IL-2-inducible T-cell kinase that causes poor E-CD4 T-cell selection due to insufficient TCR signaling improved T-CD4 T-cell generation, consistent with rescue from negative selection. Strong TCR signaling during T-CD4 T-cell development correlates with the expression of the transcription factor promyelocytic leukemia zinc finger protein. However, although modulation of the signaling strength affected the efficiency of T-CD4 T-cell development during positive and negative selection, the signaling strength is not as important for the effector function of T-CD4 T cells. These findings indicate that innate T-CD4 T cells, together with invariant natural killer T cells and γδ T cells, receive strong TCR signals during their development and that signaling requirements for the development and the effector functions are distinct.

Susceptibility to acute mouse adenovirus type 1 respiratory infection and establishment of protective immunity in neonatal mice.

There is an incomplete understanding of the differences between neonatal immune responses that contribute to the increased susceptibility of neonates to some viral infections. We tested the hypothesis that neonates are more susceptible than adults to mouse adenovirus type 1 (MAV-1) respiratory infection and are impaired in the ability to generate a protective immune response against a second infection. Following intranasal infection, lung viral loads were greater in neonates than in adults during the acute phase but the virus was cleared from the lungs of neonates as efficiently as it was from adult lungs. Lung gamma interferon (IFN-γ) responses were blunted and delayed in neonates, and lung viral loads were higher in adult IFN-γ(-/-) mice than in IFN-γ(+/+) controls. However, administration of recombinant IFN-γ to neonates had no effect on lung viral loads. Recruitment of inflammatory cells to the airways was impaired in neonates. CD4 and CD8 T cell responses were similar in the lungs of neonates and adults, although a transient increase in regulatory T cells occurred only in the lungs of infected neonates. Infection of neonates led to protection against reinfection later in life that was associated with increased effector memory CD8 T cells in the lungs. We conclude that neonates are more susceptible than adults to acute MAV-1 respiratory infection but are capable of generating protective immune responses.

Enhanced powder dispersion of dual-excipient spray-dried powder formulations of a monoclonal antibody and its fragment for local treatment of severe asthma.

The advent of biologics has brought renewed hope for patients with severe asthma, a condition notorious for being hampered by poor response to conventional therapies and adverse drug reactions owing to corticosteroid dependence. However, biologics are administered as injections, thereby precluding the benefits inhalation therapy could offer such as increased bioavailability at the site of action, minimal systemic side effects, non-invasiveness, and self-administration. Here, 2-hydroxypropyl-beta-cyclodextrin and ʟ-leucine were co-spray-dried, as protein stabiliser and dispersion enhancer, respectively, at various weight ratios to produce a series of formulation platforms. Powder aerosolisation characteristics and particle morphology were assessed for suitability for pulmonary delivery. The selected platform with the best aerosol performance, a 1:1 ratio of the excipients, was then incorporated with a monoclonal antibody directed against IL-4 receptor alpha or its antigen-binding fragment. The dual-excipient antibody formulations exhibited emitted fraction of at least 80% and fine particle fraction exceeding 60% in cascade impactor study, while the residual moisture content was within a desirable range between 1% and 3%. The in vitro antigen-binding ability and inhibitory potency of the spray-dried antibody were satisfactorily preserved. The results from this study corroborate the viability of inhaled solid-state biomacromolecules as a promising treatment approach for asthma.

Excessive amounts of mu heavy chain block B-cell development.

Antigen-independent B-cell development occurs in several stages that depend on the expression of Ig heavy and light chain. We identified a line of mice that lacked mature B cells in the spleen. This mouse line carried approximately 11 copies of a transgene of the murine heavy chain constant region locus, and B-lineage cells expressed excessive amounts of the intracellular µ heavy chain. B-cell development failed in the bone marrow at the pro/pre B-cell transition, and examination of other lines with various copy numbers of the same transgene suggested that deficiencies in B-cell development increased with increased transgene copy number. Expression of a transgenic (Tg) light chain along with the Tg µ heavy chain led to minimal rescue of B-cell development in the bone marrow and B cells in the spleen. There are several potential mechanisms for the death of pro/pre B cells as a consequence of excess heavy chain expression.

The effect of extracorporeal photopheresis on T cell response in chronic graft-versus-host disease.

Extracorporeal photopheresis (ECP) is a safe and effective immunoregulatory therapy for steroid-refractory chronic graft-versus-host disease (cGVHD) but its mechanism of action is poorly understood. In this study, we evaluated the effect of ECP in a sample of cGVHD patients. Our data showed that ECP-treated patients had lower CD4 T and B cells, and substantially higher NK cells than untreated patients. T regulatory (Treg) cells were similar between the two groups of patients. Interestingly, Treg cells were higher in ECP-treated patients and ECP-responders who had no history of aGVHD or sclerosis, than in those who had one of them or both. These findings suggest that at least one of the mechanisms of immunomodulation by ECP targets the Treg cell population and that an increase in Treg cells may be associated with response in patients with cGVHD. Together, the results of ECP are different depending on the patients' clinical condition.

IL-17 contributes to neutrophil recruitment but not to control of viral replication during acute mouse adenovirus type 1 respiratory infection.

IL-17-producing CD4(+) helper T cells (Th17 cells) promote inflammatory responses to many pathogens. We used mouse adenovirus type 1 (MAV-1) to determine contributions of IL-17 to adenovirus pathogenesis. MAV-1 infection of C57BL/6 mice upregulated lung expression of IL-17 and the Th17-associated factors IL-23 and RORγt. Only CD4(+)T cells were associated with virus-specific IL-17 production. Fewer neutrophils were recruited to airways of IL-17(-/-) mice following MAV-1 infection, but there were no other differences in pulmonary inflammation between IL-17(+/+) and IL-17(-/-) mice. Mice depleted of neutrophils using anti-Gr-1 antibody had greater lung viral loads than controls. Despite impaired neutrophil recruitment, there were no differences between IL-17(+/+) and IL-17(-/-) mice in peak lung viral loads, clearance of virus from the lungs, or establishment of protective immunity. We demonstrate robust Th17 responses during MAV-1 respiratory infection, but these responses are not essential for control of virus infection or for virus-induced pulmonary inflammation.

Prostaglandin E2 induction during mouse adenovirus type 1 respiratory infection regulates inflammatory mediator generation but does not affect viral pathogenesis.

Respiratory viruses cause substantial disease and are a significant healthcare burden. Virus-induced inflammation can be detrimental to the host, causing symptoms during acute infection and leading to damage that contributes to long-term residual lung disease. Prostaglandin E2 (PGE2) is a lipid mediator that is increased in response to many viral infections, and inhibition of PGE2 production during respiratory viral infection often leads to a decreased inflammatory response. We tested the hypothesis that PGE2 promotes inflammatory responses to mouse adenovirus type 1 (MAV-1) respiratory infection. Acute MAV-1 infection increased COX-2 expression and PGE2 production in wild type mice. Deficiency of the E prostanoid 2 receptor had no apparent effect on MAV-1 pathogenesis. Virus-induced induction of PGE2, IFN-γ, CXCL1, and CCL5 was reduced in mice deficient in microsomal PGE synthase-1 (mPGES-1(-/-) mice). However, there were no differences between mPGES-1(+/+) and mPGES-1(-/-) mice in viral replication, recruitment of leukocytes to airways or lung inflammation. Infection of both mPGES­1(+/+) and mPGES-1(-/-) mice led to protection against reinfection. Thus, while PGE2 promotes the expression of a variety of cytokines in response to acute MAV-1 infection, PGE2 synthesis does not appear to be essential for generating pulmonary immunity.

Regulation of IL-17 expression by the developmental pathway of CD4 T cells in the thymus.

CD4 T cells selected by MHC class II expressing thymocytes (T-CD4 T cells) have distinct effector functions compared to that of epithelial cell-selected CD4 T cells (E-CD4 T cells). T-CD4 T cells produce both Th1 and Th2 effector cytokines immediately after stimulation and also express IL-4 in addition to IFN-gamma under the Th1 differentiation condition. In the present study, we investigated the capability of T-CD4 T cells to become IL-17-producing cells. We found that T-CD4 T cells express reduced IL-17 under Th17-inducing conditions. T-CD4 T cells express very low levels of receptor for TGF-beta and IL-21 that are essential to induce IL-17 expression. In addition, the induction of RORgammat, a key transcription factor for IL-17 gene expression, was compromised in T-CD4 T cells under Th17 skewing conditions and ectopic expression of RORgammat restored IL-17 expression. The defect of IL-17 and RORgammat expression in T-CD4 T cells is cell intrinsic and not due to effects of a secreted factor. Thus, the developmental pathway of CD4 T cells in the thymus plays a critical role in controlling an immune response by suppressing the generation of the Th17 lineage.

Synovial autoreactive T cells in rheumatoid arthritis resist IDO-mediated inhibition.

A hallmark of T cell-mediated autoimmunity is the persistence of autoreactive T cells. However, it remains to elucidate the manner in which synovial T cells are sustained in patients with rheumatoid arthritis (RA). We found that dendritic cells (DC) and tissues from the synovial joints of RA patients expressed higher levels of IDO than DC from healthy donors. Interestingly, T cells derived from the joint synovial fluid (SF) of RA patients proliferated in response to either autologous or allogeneic IDO-positive DC, an outcome that was not affected by the addition of IDO inhibitor 1-methyl-D-tryptophan (1-MT). In contrast, addition of 1-MT to the culture stimulated with allogeneic or autologous IDO-positive DC significantly enhanced the proliferation of T cells derived from peripheral blood of healthy donors or from peripheral blood of RA patients. Furthermore, we found that functionally active tryptophanyl-tRNA-synthetase (TTS) was significantly elevated in T cells derived from the SF of RA patients, leading to enhanced storage of tryptophan in T cells and to subsequent resistance to IDO-mediated deprivation of tryptophan. The RA SF enhancement of TTS expression in T cells was blocked by mAb to IFN-gamma and TNF-alpha. These results suggest that the resistance of T cells to IDO-mediated deprivation of tryptophan represents a mechanism by which autoreactive T cells are sustained in vivo in RA patients. Specifically, blocking of the up-regulation of TTS expression in T cells presents an avenue for development of a novel therapeutic approach to treatment of RA.