Preventive azithromycin treatment reduces noninfectious lung injury and acute graft-versus-host disease in a murine model of allogeneic hematopoietic cell transplantation.

Noninfectious lung injury and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) are associated with significant morbidity and mortality. Azithromycin is widely used in allogeneic HCT recipients for pulmonary chronic GVHD, although current data appear controversial. We induced GVHD and noninfectious lung injury in lethally irradiated B6D2F1 mice by transplanting bone marrow and splenic T cells from allogeneic C57BL/6 mice. Experimental groups were treated with oral azithromycin starting on day 14 until the end of week 6 or week 14 after transplantation. Azithromycin treatment resulted in improved survival and decreased lung injury; the latter characterized by improved pulmonary function, reduced peribronchial and perivascular inflammatory cell infiltrates along with diminished collagen deposition, and a decrease in lung cytokine and chemokine expression. Azithromycin also improved intestinal GVHD but did not affect liver GVHD at week 6 early after transplantation. At week 14, azithromycin decreased liver GVHD but had no effect on intestinal GVHD. In vitro, allogeneic antigen-presenting cell (APC)- dependent T cell proliferation and cytokine production were suppressed by azithromycin and inversely correlated with relative regulatory T cell (Treg) expansion, whereas no effect was seen when T cell proliferation occurred APC independently through CD3/CD28-stimulation. Further, azithromycin reduced alloreactive T cell expansion but increased Treg expansion in vivo with corresponding downregulation of MHC II on CD11c(+) dendritic cells. These results demonstrate that preventive administration of azithromycin can reduce the severity of acute GVHD and noninfectious lung injury after allo-HCT, supporting further investigation in clinical trials.

Preventive usage of broad spectrum chemokine inhibitor NR58-3.14.3 reduces the severity of pulmonary and hepatic graft-versus-host disease.

Pulmonary graft-versus-host disease (pGVHD) is a major complication after allogeneic bone marrow transplantation (BMT), which involves donor leukocyte migration into the lung along chemokine gradients, leading to pulmonary dysfunction and respiratory insufficiency. As broad spectrum chemokine inhibitor (BSCI) NR58-3.14.3 suppresses leukocyte migration in response to various chemokines, including CCL2, CCL3, CCL5, we investigated the effects of NR58-3.14.3 on the evolution of pGVHD. Lethally irradiated B6D2F1 mice received BMT from syngeneic (B6D2F1) or allogeneic (C57BL/6) donors, and animals were treated with either NR58-3.14.3 or vehicle control from day -1 to day +14. At week 6, in allogeneic recipients that received BSCI, inflammatory cell infiltrates in the lung were decreased, and reduced histopathologic changes translated into improved pulmonary function when compared to allo-controls. Acute GVHD of the liver was also diminished, whereas no differences were seen in the gut. Alloantigen-dependent splenic T cell expansion and systemic TNF-alpha and IFN-gamma levels were comparable in NR58-3.14.3-treated animals and allo-controls. No suppressive effect of NR58-3.14.3 on CTL cytotoxicity was found, and diminished cellular infiltrates in lung and liver were most likely due to decreased migration of mononuclear cells. Therefore, novel approaches involving BSCIs may provide a promising tool in the management of pGVHD.

Pulmonary function changes in experimental graft-versus-host disease of the lung.

Pulmonary graft-versus-host disease (p-GVHD) is a serious complication after allogeneic stem cell transplantation (allo-SCT) of high morbidity and high mortality. We characterized breathing patterns and pulmonary function changes in correlation to lung histopathology and survival by using a well-established murine model of p-GVHD. Lethally irradiated B6D2F1 mice received SCT from either syngeneic B6D2F1 or allogeneic C57BL/6 animals. Within 6 weeks, severe p-GVHD developed in allogeneic recipients characterized by progressive interstitial, alveolar, peribronchial, and periluminal inflammatory cell infiltration, whereas in syngeneic recipients lung histology remained normal. Allogeneic recipients demonstrated decreased minute ventilation (MV), reduced peak inspiratory and expiratory flow rates as early as 1 week after SCT. In addition, allo-SCT resulted in restrictive pulmonary function changes as early as 7 days after transplantation and in progressive airflow obstruction within 6 weeks. Decreased breathing abilities and pulmonary function changes of allogeneic recipients were associated with increased mortality and the severity of acute graft-versus-host disease (aGVHD). These findings show that p-GVHD can be characterized by changes in pulmonary function and functional respiratory insufficiency. Furthermore, our data strengthen the understanding, that the lung is a critical target organ of aGVHD.

Oxytocin reduces anxiety via ERK1/2 activation: local effect within the rat hypothalamic paraventricular nucleus.

The neuropeptide oxytocin (OT) modulates social behaviours and is an important anxiolytic substance of the brain. However, sites of action and the intracellular signalling pathways downstream of OT receptors (OTR) within the brain remain largely unknown. In the present studies, we localized the anxiolytic effect of OT by bilateral microinfusion of OT (0.01 nmol/0.5 microL) into the hypothalamic paraventricular nucleus (PVN) in male rats using both the elevated plus-maze and the light-dark box. Moreover, intracerebroventricular administration of OT, but not of the related neuropeptide vasopressin (VP), dose-dependently activated the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade. Specifically, OT induced the phosphorylation of Raf-1, MEK1/2 and ERK1/2 in the hypothalamus in vivo and in hypothalamic H32 neurons via EGF receptors. OT-induced ERK1/2 phosphorylation was immunohistochemically localized within VP neurons of the PVN and the supraoptic nucleus. Importantly, the anxiolytic effect of OT within the PVN was prevented by local inhibition of the MAP kinase cascade with a MEK1/2 inhibitor (U0126, 0.5 nmol/0.5 microL) locally infused prior to OT, indicating the causal involvement of this intracellular signalling cascade in the behavioural effect of OT. OT effects within the hypothalamus may have far-reaching implications for the regulation of emotionality and social behaviours and, consequently, for the development of possible therapeutic strategies to treat affective disorders. Thus, OTR agonism or activation of the ERK1/2 cascade, specifically within the hypothalamus, may provide therapeutically relevant mechanisms.

Donor CD4+ T-cell production of tumor necrosis factor alpha significantly contributes to the early proinflammatory events of graft-versus-host disease.

OBJECTIVE: Tumor necrosis factor alpha (TNFalpha) is an old foe in allogeneic bone marrow transplantation (allo-BMT) promoting acute graft-versus-host disease (aGVHD). We investigated to what extent donor T cells contribute to TNFalpha production. METHODS: Lethally irradiated B6D2F1 mice were transplanted with bone marrow (BM) and T cells from syngeneic B6D2F1 or allogeneic B6 donors and assessed for cytokine production, aGVHD, and survival. RESULTS: Analysis of serum TNFalpha kinetics in recipients of allogeneic B6 wild-type BM and wild-type T cells revealed that TNFalpha levels peaked around day 7 after allo-BMT, whereas TNFalpha was undetectable in syngeneic controls. TNFalpha was produced by both host and donor cells. Further exploration showed that specifically donor CD4(+) but not CD8(+) T cells were the primary donor cell source of TNFalpha at this early time point; numbers of TNFalpha expressing splenic CD4(+) T cells were higher than CD8(+) T cells 7 days after allo-BMT, and maximal serum TNFalpha levels were detected following allo-BMT with only CD4(+) T cells compared to levels found in allogeneic recipients of only wild-type CD8(+) or to only CD4(+) TNFalpha(-/-) T cells. Concurrent with increased TNFalpha levels, early clinical aGVHD and mortality were more severe following allo-BMT with either wild-type CD4(+) and CD8(+) or CD4(+) T cells only. CONCLUSION: Our data demonstrate that in addition to residual host cells donor CD4(+) T cells significantly contribute to the proinflammatory cytokine milieu engendered early after allo-BMT through the production of TNFalpha. These findings support strategies focusing on TNFalpha neutralization as primary treatment for aGVHD.