Neutrophil-Mediated Phagocytic Host Defense Defect in Myeloid Cftr-Inactivated Mice

Cystic fibrosis (CF) is a common and deadly inherited disease, caused by mutations in the CFTR gene that encodes a cAMP-activated chloride channel. One outstanding manifestation of the disease is the persistent bacterial infection and inflammation in the lung, which claims over 90% of CF mortality. It has been debated whether neutrophil-mediated phagocytic innate immunity has any intrinsic defect that contributes to the host lung defense failure. Here we compared phagosomal CFTR targeting, hypochlorous acid (HOCl) production, and microbial killing of the neutrophils from myeloid Cftr-inactivated (Myeloid-Cftr−/−) mice and the non-inactivated control (Cftr^fl10) mice. We found that the mutant CFTR that lacked Exon-10 failed to target to the neutrophil phagosomes. This dysfunction resulted in impaired intraphagosomal HOCl production and neutrophil microbial killing. In vivo lung infection with a lethal dose of Pseudomonas aeruginosa caused significantly higher mortality in the myeloid CF mice than in the controls. The myeloid-Cftr−/− lungs were deficient in bacterial clearance, and had sustained neutrophilic inflammation and stalled transition from early to late immunity. These manifestations recapitulated the symptoms of human CF lungs. The data altogether suggest that myeloid CFTR expression is critical to normal host lung defense. CFTR dysfunction in neutrophils compromises the phagocytic innate immunity, which may predispose CF lungs to infection.     

BCL-2 is dispensable for thrombopoiesis and platelet survival

Navitoclax (ABT-263), an inhibitor of the pro-survival BCL-2 family proteins BCL-2, BCL-X_L and BCL-W, has shown clinical efficacy in certain BCL-2-dependent haematological cancers, but causes dose-limiting thrombocytopaenia. The latter effect is caused by Navitoclax directly inducing the apoptotic death of platelets, which are dependent on BCL-X_L for survival. Recently, ABT-199, a selective BCL-2 antagonist, was developed. It has shown promising anti-leukaemia activity in patients whilst sparing platelets, suggesting that the megakaryocyte lineage does not require BCL-2. In order to elucidate the role of BCL-2 in megakaryocyte and platelet survival, we generated mice with a lineage-specific deletion of Bcl2, alone or in combination with loss of Mcl1 or Bclx. Platelet production and platelet survival were analysed. Additionally, we made use of BH3 mimetics that selectively inhibit BCL-2 or BCL-X_L. We show that the deletion of BCL-2, on its own or in concert with MCL-1, does not affect platelet production or platelet lifespan. Thrombocytopaenia in Bclx-deficient mice was not affected by additional genetic loss or pharmacological inhibition of BCL-2. Thus, BCL-2 is dispensable for thrombopoiesis and platelet survival in mice.Platelets are anucleate blood cells that play essential roles in haemostasis, wound healing and a range of other processes, including inflammation and immunity.¹ They are produced by megakaryocytes, large polyploid cells that develop primarily in the bone marrow, spleen and foetal liver.² Recent work has demonstrated that the survival of megakaryocytes and platelets is governed by the BCL-2 family proteins.³ Both cell types possess a classical BAK/BAX-mediated intrinsic apoptosis pathway that must be restrained in order for them to develop and survive.In platelets, BCL-X_L is the critical pro-survival BCL-2 family member required to keep BAK and BAX in check. The first evidence of this came from Wagner et al.,⁴ who reported severe thrombocytopaenia in mice after MMTV-Cre-mediated deletion of Bclx in the haematopoietic system, skin and various secretory tissues. It has since been shown that megakaryocyte-restricted deletion of Bclx in mice reduces platelet lifespan from ~5 days to ~5 h, with a concomitant decrease in platelet counts to ~2% of wild-type levels.^{5, 6} Pharmacological inhibition of BCL-X_L with the BH3 mimetics ABT-737⁷ or Navitoclax (ABT-263)⁸ (which both also inhibit BCL-2 and BCL-W) triggers BAK/BAX-mediated platelet apoptosis.^{9, 10, 11} As a result, these drugs cause dose-dependent thrombocytopaenia in mice, dogs and humans.^{9, 11, 12, 13, 14} Indeed, thrombocytopaenia is the dose-limiting toxicity for Navitoclax.^{12, 13, 14} This fact provided additional impetus for the development of agents that specifically target BCL-2, beginning with ABT-199,¹⁵ a BCL-2-selective antagonist currently in clinical trials for the treatment of a range of haematological malignancies including chronic lymphocytic leukaemia, non-Hodgkin''s lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma and acute myeloid leukaemia. ABT-199 has already shown very promising anti-tumour activity, with little to no impact on platelet counts.^{15, 16} These data suggest that BCL-2 is dispensable for the development and survival of platelets.In megakaryocytes, BCL-X_L is also critical for survival. Although not absolutely required for their growth and maturation, BCL-X_L is essential for megakaryocytes to proceed safely through pro-platelet formation and platelet shedding.⁵ In addition to BCL-X_L, megakaryocytes also depend on the pro-survival activity of MCL-1. Conditional deletion of Mcl1 alone has no effect on this lineage. In contrast, combined megakaryocyte-specific loss of Bclx and Mcl1 results in the failure of megakaryopoiesis, systemic haemorrhage and embryonic lethality.^{5, 17, 18} These defects are rescued by deletion of Bak and Bax.¹⁸Consistent with the genetic studies, administration of ABT-737 to Mcl1^Pf4Δ/Pf4Δ mice, which lack MCL-1 in megakaryocytes and platelets, induces acute, fulminant BAK/BAX-dependent megakaryocyte apoptosis. Given that, in addition to BCL-X_L, ABT-737 also targets BCL-2,⁷ these data suggested that BCL-2 might also contribute to the development and survival of the megakaryocyte lineage. This is supported by recent studies demonstrating that neonatal human platelets contain increased levels of BCL-2 relative to adult counterparts,¹⁹ and that platelet lifespan is extended in transgenic mice expressing BCL-2 under the control of the pan-haematopoietic Vav promoter.²⁰ In light of these observations, and intense ongoing activity surrounding the development of novel BH3 mimetics,²¹ we set out to elucidate the role of BCL-2 in megakaryocytes and platelets. Mice with a megakaryocyte-specific deletion of Bcl2, either alone or in combination with deletion of Mcl1 or Bclx, were generated. The effect of these mutations, and of BCL-2 or BCL-X_L-selective BH3 mimetics, on the megakaryocyte lineage was assessed.     

Clonal CD8+ T Cell Persistence and Variable Gene Usage Bias in a Human Transplanted Hand

Immune prophylaxis and treatment of transplanted tissue rejection act indiscriminately, risking serious infections and malignancies. Although animal data suggest that cellular immune responses causing rejection may be rather narrow and predictable based on genetic background, there are only limited data regarding the clonal breadth of anti-donor responses in humans after allogeneic organ transplantation. We evaluated the graft-infiltrating CD8⁺ T lymphocytes in skin punch biopsies of a transplanted hand over 178 days. Profiling of T cell receptor (TCR) variable gene usage and size distribution of the infiltrating cells revealed marked skewing of the TCR repertoire indicating oligoclonality, but relatively normal distributions in the blood. Although sampling limitation prevented complete assessment of the TCR repertoire, sequencing further identified 11 TCR clonal expansions that persisted through varying degrees of clinical rejection and immunosuppressive therapy. These 11 clones were limited to three TCR beta chain variable (BV) gene families. Overall, these data indicate significant oligoclonality and likely restricted BV gene usage of alloreactive CD8⁺ T lymphocytes, and suggest that changes in rejection status are more due to varying regulation of their activity or number rather than shifts in the clonal populations in the transplanted organ. Given that controlled animal models produce predictable BV usage in T lymphocytes mediating rejection, understanding the determinants of TCR gene usage associated with rejection in humans may have application in specifically targeted immunotherapy.     

Age-related alterations in expression of apoptosis regulatory proteins and heat shock proteins in rat skeletal muscle

Aging of skeletal muscle is often accompanied by muscle atrophy and it appears that apoptosis plays an important role in this process. The detailed mechanism(s) is not completely understood, however. In this study, we examined expression of the apoptosis regulatory proteins as well as the heat shock proteins, which have been shown to modulate the apoptotic process in certain cell types, in order to more completely elucidate apoptotic signaling in aged skeletal muscle. To more specifically identify alterations that are likely to be the result of aging, we compared 16-month-old middle-aged (MD) and 29-month-old senescent (SE) male Fischer 344 x Brown Norway rats in our study. Our results show that the degree of DNA laddering was higher in SE compared to MD rats. Using total tissue homogenates we examined the level of expression of several apoptosis-related proteins in two categories: mitochondria-associated proteins and caspases. Of the mitochondria-associated proteins, the levels of p53 showed a significant increase in SE compared to MD rats. There was also a significant increase in the expression of Bax, Bcl-2 and Apaf-1 in SE rats over that of MD rats; cytochrome c and AIF levels remained unchanged, however. Regarding the caspases, there were increases in the levels of pro-caspases-12 and -7 and cleaved caspase-9, although the levels of pro- and cleaved caspase-3 as well as cleaved caspase-12 remained unchanged. Furthermore, our results showed significant increases in HSP27, HSP60, and the inducible HSP70. These data show that in rat skeletal muscle increased apoptosis occurs between middle-age and senescence, indicating an aging-related increase in apoptosis in skeletal muscle. The involvement of different apoptotic pathways in the aging process is suggested by the selective alterations in the apoptosis regulatory proteins. The increased expression of the HSPs suggests a relationship between HSPs and the aging-related apoptotic process.     

Simultaneous ammonium-nitrogen and copper removal, and copper recovery using nitrifying biofilm from the ultra-compact biofilm reactor

Simultaneous ammonium-nitrogen (NH(4)(+)-N) and copper removal, and copper recovery in synthetic wastewater using nitrifying biofilm from an ultra-compact biofilm reactor (UCBR) was demonstrated in batch studies, which consisted of three phases: Phase 1 for NH(4)(+)-N and copper removals, Phase 2 for copper recovery, and Phase 3 for NH(4)(+)-N removal. The results showed that more than 96.3% of copper was removed within 60min, while 60.1% of the adsorbed copper was recovered through rinsing the biofilms with 0.1mM of ethylenediaminetetraacetic acid (EDTA). The nitrifying biofilm was able to adsorb 0.245mg of copper/g of biofilms. After recovery treatment, 29.4% of copper remained bound within the nitrifying biofilms. No significant inhibitory effects towards NH(4)(+)-N removal in the presence of 0.92mg copper/L was noted in Phase 1 compared with the control test. However, lower initial pH condition in the recovery process and the accumulation of copper on the biofilm led to 50% inhibition on NH(4)(+)-N removal efficiency in the subsequent phase.