Sustained erythropoiesis and concurrent bone marrow hyperplasia are proposed to be responsible for low bone mass density (BMD) in chronic hemolytic pathologies. As impaired erythropoiesis is also frequent in these conditions, we hypothesized that free heme may alter marrow and bone physiology in these disorders. Bone status and bone marrow erythropoiesis were studied in mice with hemolytic anemia (HA) induced by phenylhydrazine (PHZ) or Plasmodium infection and in bled mice. All treatments resulted in lower hemoglobin concentrations, enhanced erythropoiesis in the spleen and reticulocytosis. The anemia was severe in mice with acute hemolysis, which also had elevated levels of free heme and ROS. No major changes in cellularity and erythroid cell numbers occurred in the bone marrow of bled mice, which generated higher numbers of erythroid blast forming units (BFU-E) in response to erythropoietin. In contrast, low numbers of bone marrow erythroid precursors and BFU-E and low concentrations of bone remodelling markers were measured in mice with HA, which also had blunted osteoclastogenesis, in opposition to its enhancement in bled mice. The alterations in bone metabolism were accompanied by reduced trabecular bone volume, enhanced trabecular spacing and lower trabecular numbers in mice with HA. Taken together our data suggests that hemolysis exerts distinct effects to bleeding in the marrow and bone and may contribute to osteoporosis through a mechanism independent of the erythropoietic stress.

Increased susceptibility to bacterial and viral infections and dysfunctional erythropoiesis are characteristic of malaria and other hemolytic hemoglobinopathies. High concentrations of free heme are common in these conditions but little is known about the effect of heme on adaptive immunity and erythropoiesis. Herein, we investigated the impact of heme (hemin) administration on immune parameters and steady state erythropoiesis in BALB/c mice, and on parasitemia and anemia during Plasmodium chabaudi adami infection. Intra-peritoneal injection of hemin (5 mg/Kg body weight) over three consecutive days decreased the numbers of splenic and bone marrow macrophages, IFN-γ responses to CD3 stimulation and Th1 differentiation. Our results show that the numbers of erythroid progenitors decreased in the bone marrow and spleen of mice treated with hemin, which correlated with reduced numbers of circulating reticulocytes, without affecting hemoglobin concentrations. Although blunted IFN-γ responses were measured in hemin-preconditioned mice, the mice developed lower parasitemia following P.c.adami infection. Importantly, anemia was exacerbated in hemin-preconditioned mice with malaria despite the reduced parasitemia. Altogether, our data indicate that free heme has dual effects on malaria pathology.

Sex differences in asthma prevalence are well-documented but poorly understood. Murine models have contributed to our understanding of mechanisms that could regulate this sex disparity, though the majority of these studies have examined responses present after Th2 adaptive immunity is established. We have now investigated how sex influences acute activation of innate cell populations in the lung upon initial exposure to the model antigen, ovalbumin (OVA), in the presence of IL-33 (OVA+IL-33), to prime the lungs for type 2 immunity. We also examined how inflammatory responses induced by OVA+IL-33 were altered in mice lacking the STAT6 transcription factor, which is activated by IL-13, an effector cytokine of IL-33. Our data demonstrate that type 2 inflammation induced by OVA+IL-33 was more severe in female mice compared to males. Females exhibited greater cytokine and chemokine production, eosinophil influx and activation, macrophage polarization to the alternatively activated phenotype, and expansion of group 2 innate lymphoid cells (ILC2s). While increases in ILC2s and eosinophils were largely independent of STAT6 in both males and females, many other responses were STAT6-dependent only in female mice. Our findings indicate that a subset of type 2 inflammatory responses induced by OVA+IL-33 require STAT6 in both males and females and that enhanced type 2 inflammation in females, compared to males, is associated with greater IL-13 protein production. Our findings suggest blunted IL-13 production in males may protect against type 2 inflammation initiated by OVA+IL-33 delivery to the lung.

Malaria is a parasitic disease that causes severe hemolytic anemia in Plasmodium-infected hosts, which results in the release and accumulation of oxidized heme (hemin). Although hemin impairs the establishment of Plasmodium immunity in vitro and in vivo, mice preconditioned with hemin develop lower parasitemia when challenged with Plasmodium chabaudi adami blood stage parasites. In order to understand the mechanism accounting for this resistance as well as the impact of hemin on eryptosis and plasma levels of scavenging hemopexin, red blood cells were labeled with biotin prior to hemin treatment and P. c. adami infection. This strategy allowed discriminating hemin-treated from de novo generated red blood cells and to follow the infection within these two populations of cells. Fluorescence microscopy analysis of biotinylated-red blood cells revealed increased P. c. adami red blood cells selectivity and a decreased permissibility of hemin-conditioned red blood cells for parasite invasion. These effects were also apparent in in vitro P. falciparum cultures using hemin-preconditioned human red blood cells. Interestingly, hemin did not alter the turnover of red blood cells nor their replenishment during in vivo infection. Our results assign a function for hemin as a protective agent against high parasitemia, and suggest that the hemolytic nature of blood stage human malaria may be beneficial for the infected host.

Type 2 immunity in the lung is promoted through the release of innate cytokines, including TSLP, from lung structural cells. These cytokines drive Type 2 immunity in part through upregulation of OX40L on dendritic cells (DCs). DCs expressing OX40L are potent inducers of Th2 differentiation. We have shown previously that STAT6 inhibitory peptide (STAT6-IP), a cell penetrating peptide designed to inhibit the STAT6 transcription factor, reduces the induction of Th2 adaptive immunity in murine models of respiratory syncytial virus infection. Here we show that intranasal administration of STAT6-IP at the time of antigen priming with ovalbumin (OVA), in conjunction with the Nod2 agonist, MDP, reduced frequencies of CD11b + lung DCs expressing OX40L. Consistent with these reductions, fewer activated DCs were localized to the lung draining lymph nodes in STAT6-IP-treated mice. Upon OVA challenge four weeks later, mice treated with STAT6-IP at the time of OVA/MDP priming did not develop airway hyperresponsiveness (AHR) and had reduced influx of eosinophils into the airways, mucus production, and serum OVA-specific IgE levels. Our findings provide evidence that the long-lasting inhibitory effects of STAT6-IP are due in part to inhibition of DC responses that drive maladaptive Th2 adaptive immunity and allergic airways disease.Keywords: Airway hyperresponsiveness r Asthma r Dendritic cells r STAT6-IP r Type 2 adaptive immunity Additional supporting information may be found online in the Supporting Information section at the end of the article.