SummaryStaphylococcal enterotoxin (SE) -induced toxic shock is triggered by inflammatory cytokine signal amplification after SE binding to major histocompatibility complex class II molecules on antigen-presenting cells and T-cell receptors. Identifying host cellular elements contributing to this pro-inflammatory signal amplification is critical for developing a strategy for therapeutic intervention. Myeloid differentiation primary-response protein 88 (MyD88) is an intracellular signalling adaptor protein primarily known for mediating pro-inflammatory cytokine responses. We investigated the role of MyD88 in staphylococcal enterotoxin A (SEA) -treated cell cultures and mouse models of toxic shock. Our results demonstrated that elevated levels of tumour necrosis factor-a, interferon-c, interleukin1a/b (IL-1a/b), IL-2 and IL-6 production correlated with up-regulation of MyD88 after treatment of spleen cells and mice with SEA alone or in combination with lipopolysaccharide (LPS). The SEA-induced lethality was also observed in (LPS-independent) D-galactosamine-sensitized mice.While LPS potentiated SEA-induced cytokine responses, D-galactosamine treatment had no additive effect. Most importantly, our results demonstrated that MyD88 )/) mice were resistant to SEA-induced toxic shock and had reduced pro-inflammatory cytokine responses. These results suggest that SEA-induced lethality is primarily dependent on MyD88. Our findings offer an important insight on potential therapeutic treatment of SEA-induced toxic shock targeting MyD88.Keywords: cytokine; D-galactosamine; knockout; lipopolysaccharides; myeloid differentiation primary-response protein 88; staphylococcal enterotoxin ANo claim to original US government works. 516Journal Compilation Ó 2010 Blackwell Publishing Ltd, Immunology, 130, 516-526 I M M U N O L O G Y O R I G I N A L A R T I C L Eon monocytes resulted in an increase in IL-1 and TNFa. 17 The SEB-dependent induction of IL-1 and TNF-a was thought to involve protein kinase C and protein tyrosine kinase. [17][18][19] Recent results indicate that IFN-c as well as IL-1 rely on myeloid differentiation factor 88 (MyD88) -dependent pathways. 20 The adaptor protein MyD88 integrates and transduces intracellular signals generated by the TLR and interleukin receptor (IL-R) superfamily, critically regulating innate immunity and host defence. 21,22 However, the impact of MyD88-mediated signalling with respect to toxic shock remains unknown.Conventionally, agonists (bacterial components) binding to host innate immune receptors, IL-1R, and all of the TLRs (excluding TLR3) prompt recruitment of the Toll-interleukin receptor (TIR) domain-containing adaptor molecule MyD88 through a TIR-TIR domain interaction. This interaction leads to downstream nuclear factor-jB (NF-jB) activation which permits the transactivation of pro-inflammatory cytokine genes. [23][24][25] With regard to TLR and MyD88 signalling in human monocytes, it has been reported that ligation of MHC class II with SEB up-regulates monocyte membrane TLR4 expressi...

An elevated pro-inflammatory cytokine response is the primary cause of death by toxic shock after exposure to staphylococcal enterotoxin B (SEB). Identifying an intracellular signal mediator that predominantly controls the pro-inflammatory response is important for developing a therapeutic strategy. We examined the role of the signaling adaptor MyD88 in cell culture and in a mouse model of toxic shock. Our results indicated that elevated tumor necrosis factor-α, interferon-γ, interleukin (IL)-1α/β and IL-6 production from mouse spleen cells treated with SEB alone or in combination with lipopolysaccharide (LPS) was regulated by MyD88. Elevated levels of MyD88 protein in spleen cells, as well as in CD11c(+) or Mac3(+) cells, and activation of nuclear factor-κB in spleen cells were observed in mice treated with SEB. An SEB-dose dependent lethality was observed in LPS-potentiated and in D-galactosamine-sensitized mice. D-Galactosamine treatment of spleen cells had no effect in cytokine induction but rather increased the sensitivity to toxic shock in mice. Our results demonstrated an impaired pro-inflammatory cytokine production by spleen cells of MyD88(-/-) mice in response to SEB or SEB plus LPS. Most importantly, MyD88(-/-) mice were resistant to SEB-induced death. These results demonstrate that MyD88-dependent pro-inflammatory signaling is responsible for SEB intoxication. In addition, our studies also demonstrated that LPS potentiation, in comparison to D-galactosamine sensitization, contributes to a stronger SEB-induced lethality. This is due to the pro-inflammatory cytokine response elicited by MyD88 after exposure to SEB and LPS. These findings offer an important insight upon SEB intoxication and subsequent therapy targeting MyD88.

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses [1][2][3] . The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system [4][5][6] . Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells 4,7,8 . These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages 9,10 . Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens 7 . While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life 6 . In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses 11 .Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-α knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice 12,13 . As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines 1,3 . Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination. Video LinkThe video component of this article can be found at https://www.jove.com/video/3960/ Protocol 1. NALT Collection and Culturing 1. Euthanize mice using approved IACUC guidance. Avoid use of inhalant anesthetics that may affect NALT. Transfer mice to an aseptic workspace or biosafety cabinet. Remove the lower jaw of the mouse and clean the upper palate area with alcohol and iodine wipes. 2. Use a No. 11 surgical blade in surgical knife handle to carefully cut and excise the upper palate by following the inside contour of the mouse incisors and molar teeth. 3. Gen...

The nasopharynx-associated lymphoid tissue (NALT) of humans and other mammals is associated with immunity against airborne infections, though it is generally considered to be a secondary component of the mucosa-associated lymphoid system. We found that protective immunity to a virulence factor of nasal mucosacolonizing Staphylococcus aureus, staphylococcal enterotoxin B (SEB), requires a functional NALT. We examined the role of NALT using intranasal (

Dengue is a mosquito-borne infection caused by four distinct serotypes of dengue virus, each appearing cyclically in the tropics and subtropics along the equator. Although vaccines are currently under development, none are available to the general population. One of the main impediments to the successful advancement of these vaccines is the lack of well-defined immune correlates of protection. Here, we describe a protein microarray approach for measuring antibody responses to the complete viral proteome comprised of the structural (capsid, membrane, and envelope) and nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) components of all four dengue virus serotypes (1 to 4). We examined rhesus macaques vaccinated with tetravalent vaccines consisting of live-attenuated virus (LAV) or purified inactivated virus (PIV), followed by boosting with LAV and challenging with wild-type dengue virus. We detected temporal increases in antibodies against envelope proteins in response to either vaccine, while only the PIV/LAV vaccination strategy resulted in anticapsid antibodies. In contrast to results from vaccination, naïve macaques challenged with wild-type viruses of each serotype demonstrated a balanced response to nonstructural and structural components, including responses against the membrane protein. Our results demonstrate discriminating details concerning the nature of antibody responses to dengue virus at the proteomic level and suggest the usefulness of this information for vaccine development. Dengue virus (DENV) is considered by the World HealthOrganization to be the greatest threat to world health among mosquito-borne viral diseases (41) and is classified by the Centers for Disease Control as a category A biothreat. Isolates of DENV are segregated into serotypes 1 to 4 (DENV-1 to DENV-4). The zoonotic transfer of dengue from sylvatic nonhuman primate hosts to sustained human transmission is estimated to have occurred between 125 and 320 years ago (38). Yellow fever, Japanese encephalitis, and West Nile viruses are other flaviviruses that are phylogenetically related to DENV and also are recently emerged human pathogens. With the exception of asymptomatic cases, the most common clinical presentation of infection by DENV is dengue fever (DF), which is characterized by acute febrile episodes and headaches, usually followed by generalized joint and muscular pain, leucopenia, and petechiae. A maculopapular rash is common in younger patients. The viremic phase of DF usually lasts for about 7 days. Although DENV serotypes 1 to 4 are all currently circulating in the world, coinfections with more than one serotype are not common. Usually resolving after a few days without major consequences, DF induces homotypic and longlasting immunity along with temporary antibody cross-reactivity against other DENV serotypes (1, 35). In contrast to DF, dengue hemorrhagic fever (DHF) is an infrequent but far more serious consequence of infection. The association between DHF and dengue virus was first described in Manila in 1953...