Modulation of inflammasome-mediated pulmonary immune activation by type I IFNs protects bone marrow homeostasis during systemic responses to Pneumocystis lung infection.

Although acquired bone marrow failure (BMF) is considered a T cell-mediated autoimmune disease, possible innate immune defects as a cause for systemic immune deviations in response to otherwise innocuous infections have not been extensively explored. In this regard, we recently demonstrated an important role of type I IFNs in protecting hematopoiesis during systemic stress responses to the opportunistic fungal pathogen Pneumocystis in lymphocyte-deficient mice. Mice deficient in both lymphocytes and type I IFN receptor (IFrag(-/-) mice) develop rapidly progressing BMF due to accelerated bone marrow (BM) cell apoptosis associated with innate immune deviations in the BM in response to Pneumocystis lung infection. However, the communication pathway between lung and BM eliciting the induction of BMF in response to this strictly pulmonary infection has been unclear. In this study, we report that absence of an intact type I IFN system during Pneumocystis lung infection not only causes BMF in lymphocyte-deficient mice but also transient BM stress in lymphocyte-competent mice. This is associated with an exuberant systemic IFN-γ response. IFN-γ neutralization prevented Pneumocystis lung infection-induced BM depression in type I IFN receptor-deficient mice and prolonged neutrophil survival time in BM from IFrag(-/-) mice. IL-1ß and upstream regulators of IFN-γ, IL-12, and IL-18 were also upregulated in lung and serum of IFrag(-/-) mice. In conjunction, there was exuberant inflammasome-mediated caspase-1 activation in pulmonary innate immune cells required for processing of IL-18 and IL-1ß. Thus, absence of type I IFN signaling during Pneumocystis lung infection may result in deregulation of inflammasome-mediated pulmonary immune activation, causing systemic immune deviations triggering BMF in this model.

Type 1 interferons suppress accelerated osteoclastogenesis and prevent loss of bone mass during systemic inflammatory responses to Pneumocystis lung infection.

HIV infection causes loss of CD4(+) T cells and type 1 interferon (IFN)-producing and IFN-responsive dendritic cells, resulting in immunodeficiencies and susceptibility to opportunistic infections, such as Pneumocystis. Osteoporosis and bone marrow failure are additional unexplained complications in HIV-positive patients and patients with AIDS, respectively. We recently demonstrated that mice that lack lymphocytes and IFN a/b receptor (IFrag(-/-)) develop bone marrow failure after Pneumocystis lung infection, whereas lymphocyte-deficient, IFN α/ß receptor-competent mice (RAG(-/-)) had normal hematopoiesis. Interestingly, infected IFrag(-/-) mice also exhibited bone fragility, suggesting loss of bone mass. We quantified bone changes and evaluated the potential connection between progressing bone fragility and bone marrow failure after Pneumocystis lung infection in IFrag(-/-) mice. We found that Pneumocystis infection accelerated osteoclastogenesis as bone marrow failure progressed. This finding was consistent with induction of osteoclastogenic factors, including receptor-activated nuclear factor-κB ligand and the proapoptotic factor tumor necrosis factor-related apoptosis-inducing ligand, in conjunction with their shared decoy receptor osteoprotegerin, in the bone marrow of infected IFrag(-/-) mice. Deregulation of this axis has also been observed in HIV-positive individuals. Biphosphonate treatment of IFrag(-/-) mice prevented bone loss and protected loss of hematopoietic precursor cells that maintained activity in vitro but did not prevent loss of mature neutrophils. Together, these data show that bone loss and bone marrow failure are partially linked, which suggests that the deregulation of the receptor-activated nuclear factor-κB ligand/osteoprotegerin/tumor necrosis factor-related apoptosis-inducing ligand axis may connect the two phenotypes in our model.

Prevention of bone marrow cell apoptosis and regulation of hematopoiesis by type I IFNs during systemic responses to pneumocystis lung infection.

We recently demonstrated that lack of type I IFN signaling (IFNAR knockout) in lymphocyte-deficient mice (IFrag(-/-)) results in bone marrow (BM) failure after Pneumocystis lung infection, whereas lymphocyte-deficient mice with intact IFNAR (RAG(-/-)) had normal hematopoiesis. In the current work, we performed studies to define further the mechanisms involved in the induction of BM failure in this system. BM chimera experiments revealed that IFNAR expression was required on BM-derived but not stroma-derived cells to prevent BM failure. Signals elicited after day 7 postinfection appeared critical in determining BM cell fate. We observed caspase-8- and caspase-9-mediated apoptotic cell death, beginning with neutrophils. Death of myeloid precursors was associated with secondary oxidative stress, and decreasing colony-forming activity in BM cell cultures. Treatment with N-acetylcysteine could slow the progression of, but not prevent, BM failure. Type I IFN signaling has previously been shown to expand the neutrophil life span and regulate the expression of some antiapoptotic factors. Quantitative RT-PCR demonstrated reduced mRNA abundance for the antiapoptotic factors BCL-2, IAP2, MCL-1, and others in BM cells from IFrag(-/-) compared with that in BM cells from RAG(-/-) mice at day 7. mRNA and protein for the proapoptotic cytokine TNF-α was increased, whereas mRNA for the growth factors G-CSF and GM-CSF was reduced. In vivo anti-TNF-α treatment improved precursor cell survival and activity in culture. Thus, we propose that lack of type I IFN signaling results in decreased resistance to inflammation-induced proapoptotic stressors and impaired replenishment by precursors after systemic responses to Pneumocystis lung infection. Our finding may have implications in understanding mechanisms underlying regenerative BM depression/failure during complex immune deficiencies such as AIDS.

Modulation of PLAGL2 transactivation by positive cofactor 2 (PC2), a component of the ARC/Mediator complex.

The pleomorphic adenoma gene (PLAG) family of transcription factors regulates a wide range of physiological processes, including cell proliferation, tissue-specific gene regulation, and embryonic development, although little is known regarding the mechanisms that regulate PLAG protein activity. In this study, a yeast two-hybrid screen identified PC2, a component of the Mediator complex, as a PLAGL2-binding protein. We show that PC2 cooperates with PLAGL2 and PU.1 to enhance the activity of a known PLAGL2 target promoter (NCF2). The PLAGL2-binding element in the NCF2 promoter consisted of the core sequence of the bipartite PLAG1 consensus site, but lacked the G-cluster motif, and was recognized by PLAGL2 zinc fingers 5 and 6. Promoter and PLAGL2 mutants showed that PLAGL2 and PU.1 were required to bind to their respective sites in the promoter, and PC2 knockdown demonstrated that PC2 was essential for enhanced promoter activity. Co-immunoprecipitation and promoter-reporter studies reveal that the effect of PC2 on PLAGL2 target promoter activity was conferred via the C-terminus of PLAGL2, the region that is required for PC2 binding and contains the PLAGL2 activation domain. Importantly, chromatin immunoprecipitation analysis and PC2 knockdown studies confirmed that endogenous PC2 protein associated with the NCF2 promoter in MM1 cells in the region occupied by PLAGL2, and was required for PLAGL2 target promoter activity in TNF-alpha-treated MM1 cells, respectively. Lastly, the expression of another known PLAGL2 target gene, insulin-like growth factor II (IGF-II), was greatly diminished in the presence of PC2 siRNA. Together, the data identify PC2 as a novel PLAGL2-binding protein and important mediator of PLAGL2 transactivation.