Timothy M. Mallers

Regulation of Apoptosis and Innate Immune Stimuli in Inflammation-Induced Preterm Labor

An innate immune response is required for successful implantation and placentation. This is regulated, in part, by the a2 isoform of V-ATPase (a2V) and the concurrent infiltration of M1 (inflammatory) and M2 (anti-inflammatory) macrophages to the uterus and placenta. The objective of the present study was to identify the role of a2V during inflammation-induced preterm labor in mice and its relationship to the regulation of apoptosis and innate immune responses. Using a mouse model of infection-induced preterm delivery, gestational tissues were collected 8 h after intrauterine inoculation on day 14.5 of pregnancy with either saline or peptidoglycan (PGN; a TLR 2 agonist) and polyinosinic-polycytidylic acid [poly(I:C); a TLR3 agonist], modeling Gram-positive bacterial and viral infections, respectively. Expression of a2V decreased significantly in the placenta, uterus, and fetal membranes during PGN+poly(I:C)-induced preterm labor. Expression of inducible NO synthase was significantly upregulated in PGN+poly(I:C)-treated placenta and uterus. PGN+poly(I:C) treatment disturbed adherens junction proteins and increased apoptotic cell death via an extrinsic pathway of apoptosis among uterine decidual cells and spongiotrophoblasts. F4/80+ macrophages were increased and polarization was skewed in PGN+poly(I:C)-treated uterus toward double-positive CD11c+ (M1) and CD206+ (M2) cells, which are critical for the clearance of dying cells and rapid resolution of inflammation. Expression of Nlrp3 and activation of caspase-1 were increased in PGN+poly(I:C)-treated uterus, which could induce pyroptosis. These results suggest that the double hit of PGN+poly(I:C) induces preterm labor via reduction of a2V expression and simultaneous activation of apoptosis and inflammatory processes.

Immune etiology of recurrent pregnancy loss and its diagnosis.

Recurrent Spontaneous Abortion of Immunological Origin (RSAI) is currently diagnosed by the occurrence of 2–3 consecutive miscarriages of unknown origin. The psychological trauma incurred by these events is a serious ailment which may be potentially avoided if a method of analysis is derived which may forecast these events and in turn prevent them from occurring. This review intends to examine studies of recurrent spontaneous abortion (RSA) which use laboratory diagnosis and also studies of RSA that do not use laboratory diagnosis. We believe that when laboratory results are incorporated into the diagnosis of RSA/RSAI that treatment is highly successful whereas the absence of laboratory results severely hinders the effectiveness of treatment. It is worth noting that correlating treatment versus outcome is imprudent because of the multiple variables involved in patient cases. It is not imprudent, however, to say that incorporation of laboratory data is essential when diagnosing RSA/RSAI.

Altered autophagic flux enhances inflammatory responses during inflammation-induced preterm labor

Cellular organelles and proteins are degraded and recycled through autophagy, a process during which vesicles known as autophagosomes fuse with lysosomes. Altered autophagy occurs in various diseases, but its role in preterm labor (PTL) is unknown. We investigated the role of autophagic flux in two mouse models of PTL compared to controls: 1) inflammation-induced PTL (IPTL), induced by toll-like receptor agonists; and 2) non-inflammation (hormonally)-induced PTL (NIPTL). We demonstrate that the autophagy related genes Atg4c and Atg7 (involved in the lipidation of microtubule-associated protein 1 light chain 3 (LC3) B-I to the autophagosome-associated form, LC3B-II) decrease significantly in uterus and placenta during IPTL but not NIPTL. Autophagic flux is altered in IPTL, as shown by the accumulation of LC3B paralogues and diminishment of lysosome associated membrane protein (LAMP)-1, LAMP-2 and the a2 isoform of V-ATPase (a2V, an enzyme involved in lysosome acidification). These alterations in autophagy are associated with increased activation of NF-κB and proinflammatory cytokines/chemokines in both uterus and placenta. Similar changes are seen in macrophages exposed to TLR ligands and are enhanced with blockade of a2V. These novel findings represent the first evidence of an association between altered autophagic flux and hyper-inflammation and labor in IPTL.

Vacuolar‐ATPase isoform a2 regulates macrophages and cytokine profile necessary for normal spermatogenesis in testis

a2V is required for maturation of sperm. The decreased expression of a2V at the feto‐maternal interphase causes poor pregnancy outcome. The present study examined the role of a2V in spermatogenesis and inflammatory network in the testis. A single dose of anti‐a2V mouse IgG or mouse IgG isotype (3 μg/animal) was injected i.p. into male mice on alternate days for 10 days. Anti‐a2V‐treated males exhibit severe deficiencies of spermatogenesis, which is indicated by the presence of less numbers of postmeiotic cells. Sperm counts and sperm motility were reduced significantly in anti‐a2V‐treated males. The release of the cleaved a2NTD was significantly lower in anti‐a2V‐treated testes. The TMs were identified as M2‐like macrophages, and this population and the expression of various cytokines/chemokines (Tgf‐β, Il‐6, Nos2, Tnf, Lif, Mcp1, Ccl5) were decreased significantly in anti‐a2V‐treated testis compared with control testis. Moreover, the cleaved a2NTD acts as a key mediator of TMs and significantly up‐regulates the secretion of testicular cytokines/chemokines, which are associated with normal spermatogenesis. When these anti‐a2V‐treated males were used for mating with normal females, the number of implantation sites was decreased significantly in the females mated with anti‐a2V‐treated males than the females mated with control males. These observations suggest that a2V plays a crucial role in spermatogenesis by regulating testicular immune responses, and its inhibition in males leads to poor pregnancy outcome in females.

Abortion‐Prone Mating Influences Alteration of Systemic a2 Vacuolar ATPase Expression in Spleen and Blood Immune Cells

a2 isoform of vacuolar ATPase (Atp6v0a2) is important for maintaining the delicate immunological balance required for successful pregnancy. The objective of this investigation is to study the dynamic changes in spleen and blood that appear during spontaneous abortion in mice.

Mouse LSTRA leukemia as a model of human natural killer T cell and highly aggressive lymphoid malignancies

Human large granular lymphocyte (LGL) leukemia is a rare lymphoproliferative disease characterized by clonal expansion of cytotoxic lymphocytes. Th ere are two major forms of human LGL leukemia: T-cell type (85%) and natural killer (NK)-cell type (15%). When NK-cell markers are coexpressed, T-LGL leukemia becomes very aggressive with a poor prognosis [1]. In patients with LGL leukemia, there is a high frequency of somatic activating mutations of signal transducer and activator of transcription 3 (STAT3) and STAT5 [2,3]. Consistent with the contribution of STAT3 to the malignant transformation of LGL, STAT3 inhibition has been shown to induce apoptosis of leukemic LGL cells from patients [4]. Th ese fi ndings strongly support the critical role

Abstract 1343: Tracking T cell leukemogenesis in mice with green fluorescence protein-labeled LSTRA leukemic cells

LSTRA leukemia was originally induced in a BALB/c mouse by the Moloney murine leukemia virus. The established LSTRA leukemic T cell line is transplantable in BALB/c mice and serves as an important animal model for leukemogenesis. Numerous studies using LSTRA cells have been reported in the field of cancer therapy, including immunotherapy and chemotherapy. Subsequent identification of a lymphocyte-specific protein tyrosine kinase gene Lck in LSTRA cells further highlights the LSTRA leukemia as a key model in studying hematologic malignancy induced by oncogenic Lck protein tyrosine kinase. While intraperitoneal inoculation of LSTRA cells reproducibly induces leukemic death in syngeneic BALB/c mice, very little is known on the details of its malignant progression. It also is not clear if LSTRA leukemia in mice resembles any specific T cell leukemia in humans. To better understand the process of LSTRA leukemia, we engineered LSTRA cells to stably express green fluorescence protein (GFP) by retroviral transduction. GFP-labeled LSTRA cells are easily distinguishable by flow cytometry, immunofluorescence microscopy, and immunoblotting. Biochemical characterization confirms that, like the parental LSTRA leukemia, GFP-tagged LSTRA cells express high levels of active Lck kinase. Consistent with our previous report (J Immunol 159:5206), GFP-tagged LSTRA cells also exhibit constitutive activation of signal transducer and activator of transcription 3 (STAT3) and STAT5. BALB/c mice receiving 10 million GFP-labeled LSTRA cells intraperitoneally show signs of leukemia, such as acute weight loss, abdominal swelling, hunched posture, and squinting eyes, within 1 week. All mice die within 10 days. Even intraperitoneal inoculation of 50 GFP-labeled LSTRA cells results in 40% lethality within a month. By tracking GFP, we were able to identify multi-organ metastasis of LSTRA leukemia, including bone marrow, spleen, and liver. Immunohistochemistry analysis of tissue sections further revealed a unique concentration of LSTRA cells in the red pulp of spleen. It is important to note that mild to moderate lymphocytic infiltration of the spleen red pulp has been reported in patients with T cell large granular lymphocyte (LGL) leukemia. It suggests that LSTRA leukemia may represent a murine model of distinct human T cell maliganacy, such as LGL leukemia. All together, we have shown here that the GFP-labeled LSTRA cell line is a powerful tool to monitor leukemic progression in immune-competent mice. It has important implications in utilizing LSTRA leukemia for future studies in cancer therapy. Experiments are also in progress to define the molecular mechanisms underlying LSTRA infiltration in the spleen red pulp. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1343. doi:1538-7445.AM2012-1343