Type I IFNs Act upon Hematopoietic Progenitors To Protect and Maintain Hematopoiesis during Pneumocystis Lung Infection in Mice.

Although acquired bone marrow failure (BMF) is considered a T cell-mediated autoimmune disease, few studies have considered contributing roles of innate immune deviations following otherwise innocuous infections as a cause underlying the immune defects that lead to BMF. Type I IFN signaling plays an important role in protecting hematopoiesis during systemic stress responses to the opportunistic fungal pathogen Pneumocystis. During Pneumocystis lung infection, mice deficient in both lymphocytes and type I IFN receptor (IFrag(-/-)) develop rapidly progressing BMF associated with accelerated hematopoietic cell apoptosis. However, the communication pathway eliciting the induction of BMF in response to this strictly pulmonary infection has been unclear. We developed a conditional-null allele of Ifnar1 and used tissue-specific induction of the IFrag(-/-) state and found that, following Pneumocystis lung infection, type I IFNs act not only in the lung to prevent systemic immune deviations, but also within the progenitor compartment of the bone marrow to protect hematopoiesis. In addition, transfer of sterile-filtered serum from Pneumocystis-infected mice as well as i.p. injection of Pneumocystis into uninfected IFrag(-/-) mice induced BMF. Although specific cytokine deviations contribute to induction of BMF, immune-suppressive treatment of infected IFrag(-/-) mice ameliorated its progression but did not prevent loss of hematopoietic progenitor functions. This suggested that additional, noncytokine factors also target and impair progenitor functions; and interestingly, fungal ß-glucans were also detected in serum. In conclusion, our data demonstrate that type 1 IFN signaling protects hematopoiesis within the bone marrow compartment from the damaging effects of proinflammatory cytokines elicited by Pneumocystis in the lung and possibly at extrapulmonary sites via circulating fungal components.

B cells modulate systemic responses to Pneumocystis murina lung infection and protect on-demand hematopoiesis via T cell-independent innate mechanisms when type I interferon signaling is absent.

HIV infection results in a complex immunodeficiency due to loss of CD4(+) T cells, impaired type I interferon (IFN) responses, and B cell dysfunctions causing susceptibility to opportunistic infections such as Pneumocystis murina pneumonia and unexplained comorbidities, including bone marrow dysfunctions. Type I IFNs and B cells critically contribute to immunity to Pneumocystis lung infection. We recently also identified B cells as supporters of on-demand hematopoiesis following Pneumocystis infection that would otherwise be hampered due to systemic immune effects initiated in the context of a defective type I IFN system. While studying the role of type I IFNs in immunity to Pneumocystis infection, we discovered that mice lacking both lymphocytes and type I IFN receptor (IFrag(-/-)) developed progressive bone marrow failure following infection, while lymphocyte-competent type I IFN receptor-deficient mice (IFNAR(-/-)) showed transient bone marrow depression and extramedullary hematopoiesis. Lymphocyte reconstitution of lymphocyte-deficient IFrag(-/-) mice pointed to B cells as a key player in bone marrow protection. Here we define how B cells protect on-demand hematopoiesis following Pneumocystis lung infection in our model. We demonstrate that adoptive transfer of B cells into IFrag(-/-) mice protects early hematopoietic progenitor activity during systemic responses to Pneumocystis infection, thus promoting replenishment of depleted bone marrow cells. This activity is independent of CD4(+) T cell help and B cell receptor specificity and does not require B cell migration to bone marrow. Furthermore, we show that B cells protect on-demand hematopoiesis in part by induction of interleukin-10 (IL-10)- and IL-27-mediated mechanisms. Thus, our data demonstrate an important immune modulatory role of B cells during Pneumocystis lung infection that complement the modulatory role of type I IFNs to prevent systemic complications.

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.

Sublingual immunization with adenovirus F protein-based vaccines stimulates protective immunity against botulinum neurotoxin A intoxication.

Sublingual (s.l.) vaccination is an efficient way to induce elevated levels of systemic and mucosal immune responses. To mediate mucosal uptake, ovalbumin (OVA) was genetically fused to adenovirus 2 fiber protein (OVA-Ad2F) to assess whether s.l. immunization was as effective as an alternative route of vaccination. Ad2F-delivered vaccines were efficiently taken up by dendritic cells and migrated mostly to submaxillary gland lymph nodes, which could readily stimulate OVA-specific CD4(+) T cells. OVA-Ad2F + cholera toxin (CT)-immunized mice elicited significantly higher OVA-specific serum IgG, IgA and mucosal IgA antibodies among the tested immunization groups. These were supported by elevated OVA-specific IgG and IgA antibody-forming cells. A mixed T(h)-cell response was induced as evident by the enhanced IL-4, IL-10, IFN-γ and TNF-α-specific cytokine-forming cells. To assess whether this approach can stimulate neutralizing antibodies, immunizations were performed with the protein encumbering the ß-trefoil domain of C-terminus heavy chain (Hcßtre) from botulinum neurotoxin A (BoNT/A) as well as when fused to Ad2F. Hcßtre-Ad2F + CT-dosed mice showed the greatest serum IgG, IgA and mucosal IgA titers among the immunization groups. Hcßtre-Ad2F alone also induced elevated antibody production in contrast to Hcßtre alone. Plasma from Hcßtre + CT- and Hcßtre-Ad2F + CT-immunized groups neutralized BoNT/A and protected mice from BoNT/A intoxication. Most importantly, Hcßtre-Ad2F + CT-immunized mice were protected from BoNT/A intoxication relative to Hcßtre + CT-immunized mice, which only showed ∼60% protection. This study shows that s.l. immunization with Ad2F-based vaccines is effective in conferring protective immunity.

Mucosal vaccine targeting improves onset of mucosal and systemic immunity to botulinum neurotoxin A.

Absence of suitable mucosal adjuvants for humans prompted us to consider alternative vaccine designs for mucosal immunization. Because adenovirus is adept in binding to the respiratory epithelium, we tested the adenovirus 2 fiber protein (Ad2F) as a potential vaccine-targeting molecule to mediate vaccine uptake. The vaccine component (the host cell-binding domain to botulinum toxin (BoNT) serotype A) was genetically fused to Ad2F to enable epithelial binding. The binding domain for BoNT was selected because it lies within the immunodominant H chain as a beta-trefoil (Hcbetatre) structure; we hypothesize that induced neutralizing Abs should be protective. Mice were nasally immunized with the Hcbetatre or Hcbetatre-Ad2F, with or without cholera toxin (CT). Without CT, mice immunized with Hcbetatre produced weak secretory IgA (sIgA) and plasma IgG Ab response. Hcbetatre-Ad2F-immunized mice produced a sIgA response equivalent to mice coimmunized with CT. With CT, Hcbetatre-Ad2F-immunized mice showed a more rapid onset of sIgA and plasma IgG Ab responses that were supported by a mixed Th1/Th2 cells, as opposed to mostly Th2 cells by Hcbetatre-dosed mice. Mice immunized with adjuvanted Hcbetatre-Ad2F or Hcbetatre were protected against lethal BoNT serotype A challenge. Using a mouse neutralization assay, fecal Abs from Hcbetatre-Ad2F or Hcbetatre plus CT-dosed mice could confer protection. Parenteral immunization showed that the inclusion of Ad2F enhances anti-Hcbetatre Ab titers even in the absence of adjuvant. This study shows that the Hcbetatre structure can confer protective immunity and that use of Hcbetatre-Ad2F gives more rapid and sustained mucosal and plasma Ab responses.