Sang-Uk Seo

Type I Interferon Signaling Regulates Ly6Chi Monocytes and Neutrophils during Acute Viral Pneumonia in Mice

Type I interferon (IFN-I) plays a critical role in the homeostasis of hematopoietic stem cells and influences neutrophil influx to the site of inflammation. IFN-I receptor knockout (Ifnar1 −/−) mice develop significant defects in the infiltration of Ly6Chi monocytes in the lung after influenza infection (A/PR/8/34, H1N1). Ly6Chi monocytes of wild-type (WT) mice are the main producers of MCP-1 while the alternatively generated Ly6Cint monocytes of Ifnar1 −/− mice mainly produce KC for neutrophil influx. As a consequence, Ifnar1 −/− mice recruit more neutrophils after influenza infection than do WT mice. Treatment of IFNAR1 blocking antibody on the WT bone marrow (BM) cells in vitro failed to differentiate into Ly6Chi monocytes. By using BM chimeric mice (WT BM into Ifnar1 −/− and vice versa), we confirmed that IFN-I signaling in hematopoietic cells is required for the generation of Ly6Chi monocytes. Of note, WT BM reconstituted Ifnar1 −/− chimeric mice with increased numbers of Ly6Chi monocytes survived longer than influenza-infected Ifnar1 −/− mice. In contrast, WT mice that received Ifnar1 −/− BM cells with alternative Ly6Cint monocytes and increased numbers of neutrophils exhibited higher mortality rates than WT mice given WT BM cells. Collectively, these data suggest that IFN-I contributes to resistance of influenza infection by control of monocytes and neutrophils in the lung.

MyD88 Signaling Is Indispensable for Primary Influenza A Virus Infection but Dispensable for Secondary Infection

ABSTRACT Recent studies have revealed that innate immunity is involved in the development of adaptive immune responses; however, its role in protection is not clear. In order to elucidate the exact role of Toll-like receptor (TLR) or RIG-I-like receptor (RLR) signaling on immunogenicity and protective efficacy against influenza A virus infection (A/PR/8/34 [PR8]; H1N1), we adapted several innate signal-deficient mice (e.g., TRIF−/−, MyD88−/−, MyD88−/− TRIF−/−, TLR3−/− TLR7−/−, and IPS-1−/−). In this study, we found that MyD88 signaling was required for recruitment of CD11b+ granulocytes, production of early inflammatory cytokines, optimal proliferation of CD4 T cells, and production of Th1 cytokines by T cells. However, PR8 virus-specific IgG and IgA antibody levels in both systemic and mucosal compartments were normal in TLR- and RLR-deficient mice. To further assess the susceptibility of these mice to influenza virus infection, protective efficacy was determined after primary or secondary lethal challenge. We found that MyD88−/− and MyD88−/− TRIF−/− mice were more susceptible to primary influenza virus infection than the B6 mice but were fully protected against homologous (H1N1) and heterosubtypic (H5N2) secondary infection when primed with a nonlethal dose of PR8 virus. Taken together, these results show that MyD88 signaling plays an important role for resisting primary influenza virus infection but is dispensable for protection against a secondary lethal challenge.

Eye Mucosa: An Efficient Vaccine Delivery Route for Inducing Protective Immunity

The external part of the eye shares mucosa-associated common characteristics and is an obvious entry site for foreign Ags. We assessed the potential of eyedrop vaccination for effective delivery of vaccines against viral or bacterial infection in mice. Both OVA-specific IgG Ab in serum and IgA Ab in mucosal compartments were induced by eyedrops of OVA with cholera toxin (CT). Eyedrop vaccination of influenza A/PR/8 virus (H1N1) induced both influenza virus-specific systemic and mucosal Ab responses and protected mice completely against respiratory infection with influenza A/PR/8 virus. In addition, eyedrop vaccination of attenuated Salmonella vaccine strains induced LPS-specific Ab and complete protection against oral challenge of virulent Salmonella. Unlike with the intranasal route, eyedrop vaccinations did not redirect administered Ag into the CNS in the presence of CT. When mice were vaccinated by eyedrop, even after the occlusion of tear drainage from eye to nose, Ag-specific systemic IgG and mucosal IgA Abs could be induced effectively. Of note, eyedrops with OVA plus CT induced organogenesis of conjunctiva-associated lymphoid tissue and increased microfold cell-like cells on the conjunctiva-associated lymphoid tissue in the nictitating membrane on conjunctiva, the mucosal side of the external eye. On the basis of these findings, we propose that the eyedrop route is an alternative to mucosal routes for administering vaccines.

Mucosa-Associated Epithelial Chemokine/CCL28 Expression in the Uterus Attracts CCR10+ IgA Plasma Cells following Mucosal Vaccination via Estrogen Control

Previous studies demonstrated cross talk between mucosal and reproductive organs during secretory IgA Ab induction. In this study, we aimed to clarify the underlying mechanisms of this cross talk. We found significantly higher titers of Ag-specific secretory IgA Ab in the vaginal wash after mucosal vaccination by both the intranasal (i.n.) and the intravaginal routes but not by the s.c. route. Interestingly, Ag-specific IgA Ab-secreting cells (ASCs) were found mainly in the uterus but not in the cervix and vaginal canal after i.n. vaccination. The fact that most Ag-specific IgA ASCs isolated from the uteri of vaccinated mice migrated toward mucosa-associated epithelial chemokine (MEC)/CCL28 suggests dominant expression of CCR10 on the IgA ASCs. Further, IgA ASCs in the uteri of vaccinated mice were reduced drastically in mice treated with neutralizing anti-MEC/CCL28 Ab. Most intriguingly, the female sex hormone estrogen directly regulated MEC/CCL28 expression and was augmented by i.n. vaccination with cholera toxin or stimulators for innate immunity. Further, blockage of estrogen function in the uterus by oral administration of the estrogen antagonist raloxifene significantly inhibited migration of Ag-specific IgA ASCs after i.n. vaccination with OVA plus cholera toxin. Taken together, these data strongly suggest that CCR10+ IgA ASCs induced by mucosal vaccination via the i.n. route migrate into the uterus in a MEC/CCL28-dependent manner and that estrogen might have a crucial role in the protection against genital infection by regulating MEC/CCL28 expression in the uterus.

Interleukin-1 Promotes Coagulation, Which Is Necessary for Protective Immunity in the Lung Against Streptococcus pneumoniae Infection

Interleukin (IL)-1 is a well-known cytokine for the initiation of innate immunity in bacterial infection. However, the underlying mechanism of IL-1 on the respiratory infection is not fully elucidated. We studied how IL-1 contributes to the host defense against Streptococcus pneumoniae. IL-1R(-/-) mice showed high mortality, local cytokine storm, and substantial infiltrates in the lower respiratory tract after intratracheal challenge with S. pneumoniae. The IL-1-deficient condition did not suppress the propagation of bacteria in the lung, although the recruitment and the bacteria-killing ability of neutrophils (CD11b(+)Ly6C(+)Ly6G(+)) were not defective compared with wild-type mice. Unexpectedly, we found that the transcription of fibrinogen alpha and gamma genes were highly activated in the lungs of wild-type mice after the infection, whereas no significant changes were found in IL-1R(-/-) mice. Of note, synthesis of fibrinogen was dependent on the IL-1-IL-6-Stat3 cascade. Treatment with recombinant fibrinogen improved survival and bacterial propagation in the IL-1R(-/-) mice and blockade of the coagulation increased the susceptibility of wild-type mice to pneumococcal pneumonia. Our findings suggest that IL-1 signaling leads to the synthesis of fibrinogen in the lung after pneumococcus infection and is followed by coagulation, which contributes to the control of bacterial infection in the pulmonary tract.

Expansion of Tfh‐like cells during chronic Salmonella exposure mediates the generation of autoimmune hypergammaglobulinemia in MyD88‐deficient mice

The role of TLR signaling in linking the innate and adaptive immune systems has been a controversial issue that remains to be solved. Here, we determined whether MyD88‐dependent TLR signals are required for the generation of B‐cell responses during chronic Salmonella infection. Oral administration of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine (RASV) strain in MyD88−/− mice resulted in chronic infection. Infection was accompanied by enlarged germinal centers and hypergammaglobulinemia with anti‐double‐stranded DNA (dsDNA)‐specific Ab in sera, and the deposition of immune complexes in the kidneys, suggesting onset of autoimmunity. CD4+ T cells expressing PD‐1, CXCR5, ICOS, and IL‐21 were dramatically increased in chronically infected mice, indicating the expansion of follicular helper T (Tfh)‐like cells. Of note, the depletion of CD4+ T cells completely blocked the generation of polyclonal IgG Ab in sera after oral RASV challenge. Inflammatory myeloid cells expressing CD11b and Gr‐1 accumulated in high numbers in the spleen of MyD88−/− mice. Interestingly, the blockade of PD‐1 or ICOS significantly reduced the hypergammaglobulinemia and dsDNA‐specific autoantibody production. Overall, these results suggest that Tfh‐like cells in chronic bacterial infection trigger autoimmune hypergammaglobulinemia in a PD‐1‐ and ICOS‐dependent manner.

Development and characterization of a live attenuated influenza B virus vaccine candidate.

A human influenza B/Lee/40 virus was cold-adapted by serial passages in embryonated chicken eggs, at progressively lower temperatures, for possible use as a future influenza B vaccine donor strain. Temperature sensitive and cold-adapted phenotypes were achieved as a consequence of the adaptation process. It was determined that the virus was attenuated in mice since the replication of the viral genome was significantly reduced in the lung. Despite decreased viral replication, the attenuated infection effectively induced a virus-specific immune response. We next developed a reassortant virus carrying two major surface proteins, hemagglutinin and neuraminidase from virulent B/Shangdong/7/97 and six internal genes from the cold-adapted B/Lee/40. The reassortant virus was also attenuated and protected mice from lethal challenge with wild type B/Shangdong/7/97. In addition, vaccination with the reassortant virus resulted in a specific antibody response and inhibited the replication of wild type virus in mice. We conclude that the cold-adapted B/Lee/40 donor strain merits further investigation as potential live vaccine carrier as an alternative means for protection from influenza B virus epidemics.

Effective protection against secondary pneumococcal pneumonia by oral vaccination with attenuated Salmonella delivering PspA antigen in mice

Influenza infection followed by pneumococcal infection can cause severe pneumonia and this secondary pneumococcal pneumonia is the most common cause of influenza-associated death. Therefore, vaccination against Streptococcus pneumoniae is highly desirable for reducing the disease burden caused by seasonal epidemic and pandemic influenza. In this study, mice were vaccinated orally with a recombinant attenuated Salmonella vaccine (RASV) strain that delivers pneumococcal surface protein A (PspA) in order to examine protective efficacy against secondary pneumococcal pneumonia. A single dose of oral RASV resulted in attenuated pulmonary inflammation and effective protection against secondary pneumococcal pneumonia. Additionally, oral RASV induced long-term protection against secondary pneumococcal pneumonia. Treatment with an antiviral drug (i.e., oseltamivir) after treatment with RASV further prevented pulmonary inflammation after secondary pneumococcal infection. These results imply that oral RASV can protect mice from secondary pneumococcal infection after influenza infection and that vaccination against respiratory bacterial pathogens is a promising approach for dampening the impact of annual epidemic and pandemic influenza outbreaks.

IFN-γ Secreted by CD103+ Dendritic Cells Leads to IgG Generation in the Mesenteric Lymph Node in the Absence of Vitamin A

Although the induction mechanism of secretory IgA has been well studied, that of IgG in the mucosal compartments is not well understood. In this study, vitamin A deficiency was convincingly shown to be associated with increased IgG in serum and intestinal fluid. We found increased numbers of IgG-secreting B cells in the lamina propria of the small intestine and mesenteric lymph node (MLN) of vitamin A-deficient (VAD) mice. Of note, IFN-γ secreted by MLN dendritic cells (DCs) was significantly augmented in VAD mice, unlike control mice, and CD103+ DCs were the main subsets to secrete IFN-γ. The aberrant increase of IgG in VAD mice can be ascribable to IFN-γ, because IFN-γ−/− VAD mice have normal IgG levels and the addition of rIFN-γ increased IgG production by B cells cocultured with MLN DCs from IFN-γ−/− VAD mice. Oral feeding of antibiotics resulted in significant reduction of IgG in VAD mice, indicating a critical role for altered commensal bacteria for IgG class-switching recombination in the absence of vitamin A. Collectively, vitamin A deficiency provokes the generation of IFN-γ–secreting CD103+ DCs, which may be a critical regulator for IgG generation in the MLN.

Genotyping and screening of reassortant live-attenuated influenza B vaccine strain

Live-attenuated influenza virus vaccines can be generated by reassortment of gene segments between an attenuated donor strain and a virulent wild-type virus. The annual production schedule for the seasonal influenza vaccine necessitates rapid and efficient genotyping of the reassorted progeny to identify the desired vaccine strains. This study describes a multiplex RT-PCR system capable of identifying each gene segment from the cold-adapted attenuated donor virus, B/Lee/40ca. The specificity of the amplification system was optimized by testing various wild-type influenza B viruses. The resulting RT-PCR method is sensitive and efficient enough for routine identification of reassortant clones to identify the desired gene constellation, consisting of six segments from the attenuated donor virus and the H and N genes from the wild-type virus. By providing a more rapid and efficient means of genotyping the candidate reassortant strains, this method could be implemented to expedite the generation of each component strain and allow more time to culture and process the final seasonal influenza vaccine.