Nicholas M. Ponzio

Cytokine Levels during Pregnancy Influence Immunological Profiles and Neurobehavioral Patterns of the Offspring

Abstract:  The underlying causes of autism spectrum disorders (ASD) are unknown, but clinical and experimental studies indicate immune mechanisms, in general, and cytokine dysregulation, in particular, as contributing factors in their etiology. We developed a prenatal mouse model of autism to demonstrate that circulating levels of defined cytokines in pregnant dams could influence fetal development and behavioral characteristics in their offspring. We administered daily injections of murine IL‐2 (0.4 μg in phosphate‐buffered saline [PBS]) to pregnant mice during mid‐gestation, and analyzed their offspring (IL‐2 pups) in comparison to offspring of pregnant mice injected with vehicle only (PBS pups). Significant levels of IL‐2 were present in amniotic fluid and tissues from embryos of dams given radiolabeled IL‐2, indicating that the injected IL‐2 crossed the placenta and entered the fetuses. Lymphocytes from IL‐2 pups demonstrated accelerated T cell development, with a skewing toward TH1 cell differentiation. IL‐2 pups also showed in vitro proliferative and cytotoxicity responses that were significantly higher than control PBS pups when stimulated with syngeneic B lymphoma cells or allogeneic spleen cells. In addition to their previously shown increases in open‐field activity, grooming and rearing behavior, offspring of IL‐2‐injected (vs. PBS‐injected) dams also displayed abnormal new motor learning as assessed through acquisition of the classically conditioned eyeblink response. These results suggest that increases in maternal levels of IL‐2 during pregnancy induce in their offspring long‐lasting increased vulnerability to neurobehavioral abnormalities associated with autism, and provide a valid animal model to determine the underlying immunological mechanisms.

Mesenchymal Stem Cell–Derived Exosomes Stimulate Cycling Quiescence and Early Breast Cancer Dormancy in Bone Marrow

Dormant breast cancers resurge as metastatic disease after a long dormancy period in the bone marrow, where cancer cells interact with mesenchymal stem cells (MSC). However, the nature of early interactions between breast cancer cells and MSCs in the bone marrow microenvironment that facilitate adaptation to a quiescent state remains poorly understood. Here, we report that breast cancer cells prime MSC to release exosomes containing distinct miRNA contents, such as miR-222/223, which in turn promotes quiescence in a subset of cancer cells and confers drug resistance. Building on these results, we developed a novel, nontoxic therapeutic strategy to target dormant breast cancer cells based on systemic administration of MSC loaded with antagomiR-222/223. In an immunodeficient mouse model of dormant breast cancer, this therapy sensitized breast cancer cells to carboplatin-based therapy and increased host survival. Overall, our findings illuminate the nature of the regulatory interactions between breast cancer cells and MSCs in the evolution of tumor dormancy and resurgence in the micrometastatic microenvironment of the bone marrow. Cancer Res; 76(19); 5832-44. ©2016 AACR.

IL-10 production in a CD5+ B cell lymphoma arising in a CD4 monoclonal antibody-treated SJL mouse.

A majority of SJL mice develop spontaneous reticulum cell sarcomas (RCS) at about 1 year of age which can be transplanted into young SJL recipients. Previous studies have shown that RCS tumors are of B cell lineage, and that the development of these lymphomas and their subsequent growth depends upon host-derived T helper cell factors. In vivo treatment of SJL mice with anti-CD4 monoclonal antibody (mAb) prevents the development of the characteristic B lymphomas. Most of the mAb-treated animals were tumor free and had a significantly prolonged life span. However, one such CD4 mAb-treated mouse developed a transplantable IgM+ CD5+ B cell lymphoma (designated NJ101), which has not previously been described in SJL/J mice. NJ101 is clonal on the basis of a discrete non-germ line Ig heavy chain gene rearrangement by Southern blot analysis. Unlike the sIg- CD5- transplantable RCS-X cell line, the IgM+ CD5+ NJ101 lymphoma cells will grow in immuno-compromised hosts, such as irradiated recipients or in recipients treated with CD4 mAb in vivo. The RCS (B cell) lymphoma requires CD4+ T cells for progressive growth, whereas the growth of the CD5+ B lymphoma cells is enhanced by the removal of such cells. Thus, CD5+ B cell clonal development may be aided by the removal of regulatory T cells and/or the malignant CD5+ B cells may produce their own growth factors in an autocrine manner. Examination of IL-10 message by quantitative polymerase chain reaction techniques indicate that the CD5+ B lymphoma cells produce increased levels of IL-10 relative to normal LN cells or purified RCS lymphoma cells. These results suggest that two different types of B cell tumors, both of which can develop in SJL mice, have different growth requirements. Furthermore, treatment to prevent the occurrence of the characteristic RCS malignancy of SJL mice may lead to the development of another form of B cell neoplasia.

B cell lymphomas of C57l/J mice; the role of natural killer cells and T helper cells in lymphoma development and growth

The Hodgkins-like Type B neoplasms which arise spontaneously in aging C57L mice (25% incidence at 21 months of age) were first reported over 40 years ago, but since then relatively little has been published about these lymphomas. Based on previous studies in SJL mice, we investigated the phenotypic and functional properties of C57L-derived lymphomas in relation to Mtv29-encoded vSAg expression by the tumor cells, and their ability to stimulate TCR Vbeta-restricted T cells. The cell surface phenotype of the C57L lymphomas indicates a B cell origin (sIg(+), MHC II(+)). These B lymphoma cells also express co-stimulatory molecules [B7-1 (CD80) and HSA (CD24)], and stimulate marked proliferation of syngeneic CD4(+) T cells. C57L B lymphoma cells exhibit Mtv-encoded mRNA by northern analysis, and also stimulate IL-2 production from Vbeta16(+) T cell hybrids, suggesting a role for Mtv 29 in this syngeneic T cell response. After transfer to syngeneic recipients, primary C57L lymphomas grow slowly, if at all. However, tumor growth is greatly accelerated by pretreatment of C57L recipients with anti-asialo GM1 antibody (but not anti-CD8 mAb), suggesting that NK cells play a major role in inhibiting lymphoma growth. If, in addition to anti-asialo GM1, the mice are also pretreated with anti-CD4 mAb, tumor growth is markedly inhibited, indicating that the lymphoma-responsive syngeneic CD4(+) T cells promote tumor growth. Therefore, although the vSAg-induced response stimulated by vSAg29 expressing lymphoma cells in syngeneic TCR Vbeta-restricted CD4(+) T cells is an important etiologic factor in this type of B cell neoplasm both in C57L and in SJL mice, the final outcome of the spontaneous neoplastic process appears strongly influenced by endogenous NK activity in aging mice.

Long-Term Exposure of HL60 Cells to 1,25-Dihydroxyvitamin D3 Reduces Their Tumorigenicity: A Model for Cancer Chemoprevention

Abstract Several lines of evidence suggest that 1,25-dihydroxyvitamin D3 (1,25D3) may be important in chemoprevention of human cancer. Here, we show that human promyelocytic leukemia cells HL60 cultured in the presence of 30 nM 1,25D3 (30A cells) for 3 years exhibited a reduced rate of tumor growth when injected into nu/nu mice, while cells grown in 40 nM 1,25D3 (40AF cells) failed to form detectable tumors in 11 out of the 12 inoculated mice. Interestingly, both 30A and 40AF cells grew approximately twice as fast as the parental HL60-G cells under tissue culture conditions, even in the presence of 1,25D3, to which they developed resistance. Tests of the susceptibility of these cells to natural killer (NK) cell cytotoxicity showed that 40AF, but not HL60-G or 30A cells, were targets for the murine spleen NK cells. However, lysis of 30A cells was also detected when human NK cells were used in this assay, though the effector-to-target cell ratio necessary to obtain significant lysis above background levels was higher for 30A (80:1) than for 40AF (10:1) cells. These results suggest a mechanism for the reported chemopreventive effects of sunlight-generated 1,25D3 or dietary vitamin D3.

Immunostimulatory effects of mesenchymal stem cell-derived neurons: implications for stem cell therapy in allogeneic transplantations.

Mesenchymal stem cells (MSCs) differentiate along various lineages to specialized mesodermal cells and also transdifferentiate into cells such as ectodermal neurons. MSCs are among the leading adult stem cells for application in regenerative medicine. Advantages include their immune‐suppressive properties and reduced ethical concerns. MSCs also show immune‐enhancing functions. Major histocompatibility complex II (MHC‐II) is expected to be downregulated in MSCs during neurogenesis. Ideally, “off the shelf” MSCs would be suited for rapid delivery into patients. The question is whether these MSC‐derived neurons can reexpress MHC‐II in a milieu of inflammation. Western analyses demonstrated gradual decrease in MHC‐II during neurogenesis, which correlated with the expression of nuclear CIITA, the master regulator of MHC‐II expression. MHC‐II expression was reversed by exogenous IFNY. One‐way mixed lymphocyte reaction with partly differentiated neurons showed a stimulatory effect, which was partly explained by the release of the proinflammatory neurotransmitter substance P (SP), cytokines, and decreases in miR‐130a and miR‐206. The anti‐inflammatory neurotransmitters VIP and CGRP were decreased at the peak time of immune stimulation. In summary, MSC‐derived neurons show decreased MHC‐II expression, which could be reexpressed by IFNY. The release of neurotransmitters could be involved in initiating inflammation, underscoring the relevance of immune responses as consideration for stem cell therapies.

Superantigens related to B cell hyperplasia

ConclusionsIn both mouse and humans, B cell hyperplasia is seen in many situations that involve T cell stimulation. In the mouse, some of these conditions appear to involve a SAg-induced T cell response, which can result in chronic B cell hyperplasia and development of B cell lymphomas. From our investigation of the B cell lymphomas that characteristically arise in a majority (≃90%) of SJL/J mice, it is clear that a vSAg which is encoded by a unique endogenous mammary tumor provirus is involved in the neoplastic process. The tumor-associated vSAg (mtv-29-LTR) stimulates a TCR Vβ-restricted subset of T cells (Vβ16+) to produce cytokines which the lymphoma cells use as paracrine growth factors for progressive growth. We speculate that themtv-29-encoded vSAg may be expressed on germinal center B cells, even in young SJL mice, and that the characteristic chronic B cell hyperplasia seen in this strain is driven by the cytokines produced by responding Vβ16+ T cells. Moreover, the cytokine dependence of the SJL B cell lymphomas on IL-5 and IL-4 suggests that preferential stimulation of Th2 cells is involved. This scenario of B cell hyperplasia associated with SAg-induced Th2 cell stimulation may also be a major element in other murine models, such as chronic parent → F1 GvHD and MAIDS.Mechanisms similar to those described in these mouse models may also be operative in human diseases caused by infectious microorganisms, such asH. pylori, EBV, HIV,M. arthritidis, mycobacteria, malarial parasites, and others. However, the involvement of SAgs in these conditions, and their precise role in eliciting from T cells those eytokines which promote chronic B cell hyperplasia and lymphoma development remain to be clearly defined. The possible role ofγδ T cells in similar disease mechanisms will need to be explored in the light of overwhelming evidence that certain unconventional antigens such as HSP stimulate subpopulations of these T cells. In our discussion of these diseases we have, therefore, given consideration to the possibility that the definition of SAg may have to be expanded to include such antigens.

Pharmacokinetics and modeling of immune cell trafficking: quantifying differential influences of target tissues versus lymphocytes in SJL and lipopolysaccharide-treated mice

BackgroundImmune cell trafficking into the CNS and other tissues plays important roles in health and disease. Rapid quantitative methods are not available that could be used to study many of the dynamic aspects of immune cell-tissue interactions.MethodsWe used pharmacokinetics and modeling to quantify and characterize the trafficking of radioactively labeled lymphocytes into brain and peripheral tissues. We used variance from two-way ANOVAs with 2 × 2 experimental designs to model the relative influences of lymphocytes and target tissues in trafficking.ResultsWe found that in male CD-1 mice, about 1 in 5,000 intravenously injected lymphocytes entered each gram of brain. Uptake by brain was 2 to 3 times higher in naïve SJL females, but uptake by spleen and clearance from blood was lower, demonstrating a dichotomy in immune cell distribution. Treatment of CD-1 mice with lipopolysaccharide (LPS) increased immune cell uptake into brain but decreased uptake by spleen and axillary nodes.ConclusionsDifferences in brain uptake and in uptake by spleen between SJL and CD-1 mice were primarily determined by lymphocytes, whereas differences in uptake with LPS were primarily determined by lymphocytes for the brain but by the tissues for the spleen and the axillary lymph node. These results show that immune cells normally enter the CNS and that tissues and immune cells interact in ways that can be quantified by pharmacokinetic models.

Murine survival of lethal irradiation with the use of human umbilical cord blood

We have found that human umbilical cord blood (HUCB) will routinely protect mice exposed to lethal levels of irradiation. At the end of 50 days, over seventy percent (70%) of mice injected with HUCB survived 900 cGy or irradiation, which produced 100% deaths in the uninjected control animals. Moreover, there was some evidence that human colony stimulating factors further improved survival. Anti-Natural Killer cell (NK) antibody was utilized along with HUCB in these studies, however, Anti-NK cell serum alone had no radioprotective effect in mice. The studies reported here suggest the possibility of utilizing HUCB for immediate protection of humans from lethal irradiation.

The maternal immune system during pregnancy and its influence on fetal development

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