Concurrent Brain Subregion Microgliosis in an HLA-II Mouse Model of Group A Streptococcal Skin Infection.

The broad range of clinical manifestations and life-threatening infections caused by the Gram-positive bacterium, Streptococcus pyogenes or Group A Streptococcus (GAS), remains a significant concern to public health, with a subset of individuals developing neurological complications. Here, we examined the concurrent neuroimmune effects of subcutaneous GAS infections in an HLA-Class II (HLA) transgenic mouse model of subcutaneous GAS infection. To investigate changes in the skin-brain axis, HLA-DQ8 (DQA1*0301/DQB1*0302) mice (DQ8) were randomly divided into three groups: uninfected controls (No Inf), GAS infected and untreated (No Tx), and GAS infected with a resolution by clindamycin (CLN) treatment (CLN Tx) (10 mg/kg/5 days) and were monitored for 16 days post-infection. While the skin GAS burden was significantly reduced by CLN, the cortical and hippocampal GAS burden in the male DQ8 mice was not significantly reduced with CLN. Immunoreactivity to anti-GAS antibody revealed the presence of GAS bacteria in the vicinity of the neuronal nucleus in the neocortex of both No Tx and CLN Tx male DQ8 mice. GAS infection-mediated cortical cytokine changes were modest; however, compared to No Inf or No Tx groups, a significant increase in IL-2, IL-13, IL-22, and IL-10 levels was observed in CLN Tx females despite the lack of GAS burden. Western blot analysis of cortical and hippocampal homogenates showed significantly higher ionized calcium-binding adaptor-1 (Iba-1, microglia marker) protein levels in No Tx females and males and CLN Tx males compared to the No Inf group. Immunohistochemical analysis showed that Iba-1 immunoreactivity in the hippocampal CA3 and CA1 subregions was significantly higher in the CLN Tx males compared to the No Tx group. Our data support the possibility that the subcutaneous GAS infection communicates to the brain and is characterized by intraneuronal GAS sequestration, brain cytokine changes, Iba-1 protein levels, and concurrent CA3 and CA1 subregion-specific microgliosis, even without bacteremia.

HLA-II-Dependent Neuroimmune Changes in Group A Streptococcal Necrotizing Fasciitis.

Streptococcus pyogenes (Group A Streptococcus, GAS) bacteria cause a spectrum of human diseases ranging from self-limiting pharyngitis and mild, uncomplicated skin infections (impetigo, erysipelas, and cellulitis) to highly morbid and rapidly invasive, life-threatening infections such as streptococcal toxic shock syndrome and necrotizing fasciitis (NF). HLA class II allelic polymorphisms are linked with differential outcomes and severity of GAS infections. The dysregulated immune response and peripheral cytokine storm elicited due to invasive GAS infections increase the risk for toxic shock and multiple organ failure in genetically susceptible individuals. We hypothesized that, while the host immune mediators regulate the immune responses against peripheral GAS infections, these interactions may simultaneously trigger neuropathology and, in some cases, induce persistent alterations in the glial phenotypes. Here, we studied the consequences of peripheral GAS skin infection on the brain in an HLA-II transgenic mouse model of GAS NF with and without treatment with an antibiotic, clindamycin (CLN). Mice expressing the human HLA-II DR3 (DR3) or the HLA-II DR4 (DR4) allele were divided into three groups: (i) uninfected controls, (ii) subcutaneously infected with a clinical GAS strain isolated from a patient with GAS NF, and (iii) GAS-infected with CLN treatment (10 mg/kg/5 days, intraperitoneal). The groups were monitored for 15 days post-infection. Skin GAS burden and lesion area, splenic and hippocampal mRNA levels of inflammatory markers, and immunohistochemical changes in hippocampal GFAP and Iba-1 immunoreactivity were assessed. Skin GAS burden and hippocampal mRNA levels of the inflammatory markers S100A8/A9, IL-1ß, IL-33, inflammasome-related caspase-1 (Casp1), and NLRP6 were elevated in infected DR3 but not DR4 mice. The levels of these markers were significantly reduced following CLN treatment in DR3 mice. Although GAS was not detectable in the brain, astrocyte (GFAP) and microglia (Iba-1) activation were evident from increased GFAP and Iba-1 mRNA levels in DR3 and DR4 mice. However, CLN treatment significantly reduced GFAP mRNA levels in DR3 mice, not DR4 mice. Our data suggest a skin-brain axis during GAS NF, demonstrating that peripherally induced pathological conditions regulate neuroimmune changes and gliotic events in the brain.

Effects of Probiotics on Colitis-Induced Exacerbation of Alzheimer's Disease in AppNL-G-F Mice.

Alzheimer's disease (AD) is characterized by progressive cognitive decline and is a leading cause of death in the United States. Neuroinflammation has been implicated in the progression of AD, and several recent studies suggest that peripheral immune dysfunction may influence the disease. Continuing evidence indicates that intestinal dysbiosis is an attribute of AD, and inflammatory bowel disease (IBD) has been shown to aggravate cognitive impairment. Previously, we separately demonstrated that an IBD-like condition exacerbates AD-related changes in the brains of the AppNL-G-F mouse model of AD, while probiotic intervention has an attenuating effect. In this study, we investigated the combination of a dietary probiotic and an IBD-like condition for effects on the brains of mice. Male C57BL/6 wild type (WT) and AppNL-G-F mice were randomly divided into four groups: vehicle control, oral probiotic, dextran sulfate sodium (DSS), and DSS given with probiotics. As anticipated, probiotic treatment attenuated the DSS-induced colitis disease activity index in WT and AppNL-G-F mice. Although probiotic feeding significantly attenuated the DSS-mediated increase in WT colonic lipocalin levels, it was less protective in the AppNL-G-F DSS-treated group. In parallel with the intestinal changes, combined probiotic and DSS treatment increased microglial, neutrophil elastase, and 5hmC immunoreactivity while decreasing c-Fos staining compared to DSS treatment alone in the brains of WT mice. Although less abundant, probiotic combined with DSS treatment demonstrated a few similar changes in AppNL-G-F brains with increased microglial and decreased c-Fos immunoreactivity in addition to a slight increase in Aß plaque staining. Both probiotic and DSS treatment also altered the levels of several cytokines in WT and AppNL-G-F brains, with a unique increase in the levels of TNFα and IL-2 being observed in only AppNL-G-F mice following combined DSS and probiotic treatment. Our data indicate that, while dietary probiotic intervention provides protection against the colitis-like condition, it also influences numerous glial, cytokine, and neuronal changes in the brain that may regulate brain function and the progression of AD.

HLA-II Alleles Influence Physical and Behavioral Responses to a Whey Allergen in a Transgenic Mouse Model of Cow's Milk Allergy.

The symptoms of food allergies vary significantly between individuals, likely due to genetic determinants. In humans, allergy development is initiated by antigen-presenting cells via class II human leukocyte antigen (HLA-II). The HLA-II gene is highly polymorphic, and its allelic variance is thought to influence the susceptibility of individuals to a particular allergen. However, whether antigen presentation by different HLA-II variants contributes to symptom variation is not clear. We hypothesized that HLA-II allelic variance affects symptom phenotypes, including immediate physical reactions and delayed behavioral changes, in individuals with food hypersensitivity. To test our hypothesis, male and female mice of three transgenic strains expressing an HLA-II variant, DR3, DR15, or DQ8, were used to establish a cow's milk allergy model. Mice were sensitized to a bovine whey allergen, ß-lactoglobulin (BLG; Bos d 5), weekly for 5 weeks, followed by an acute oral allergen challenge. At 30 min post-challenge, BLG-sensitized DR3 mice showed moderate to severe anaphylaxis resulting in perioral redness, swelling, and death. In contrast, DQ8 and DR15 mice were generally asymptomatic. The production of allergen-specific immunoglobulins was also HLA- and sex-dependent. Both male and female DR3 and female DR15 mice significantly increased BLG-specific IgE production, while robust elevation in BLG-specific IgG1 was observed in sensitized DQ8 mice of both sexes and, to a lesser extent, in DR15 males. Furthermore, BLG-sensitized DR15 mice showed sex-specific behavior changes, with males exhibiting mobility changes and anxiety-like behavior and females showing spatial memory impairment. When splenocytes from transgenic mice were stimulated in vitro with BLG, phenotypes of immune cells were HLA- and sex-specific, further underscoring the influence of HLA-II on immune responses. Our results support that HLA-II alleles influence behavioral responses in addition to immune and physical reactions of food allergy, suggesting that certain HLA-II variants may predispose individuals to food-allergy-associated behavioral changes.

Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity.

Non-canonical inflammasome activation by mouse caspase-11 (or human CASPASE-4/5) is crucial for the clearance of certain gram-negative bacterial infections, but can lead to severe inflammatory damage. Factors that promote non-canonical inflammasome activation are well recognized, but less is known about the mechanisms underlying its negative regulation. Herein, we identify that the caspase-11 inflammasome in mouse and human macrophages (Mϕ) is negatively controlled by the zinc (Zn2+) regulating protein, metallothionein 3 (MT3). Upon challenge with intracellular lipopolysaccharide (iLPS), Mϕ increased MT3 expression that curtailed the activation of caspase-11 and its downstream targets caspase-1 and interleukin (IL)-1ß. Mechanistically, MT3 increased intramacrophage Zn2+ to downmodulate the TRIF-IRF3-STAT1 axis that is prerequisite for caspase-11 effector function. In vivo, MT3 suppressed activation of the caspase-11 inflammasome, while caspase-11 and MT3 synergized in impairing antibacterial immunity. The present study identifies an important yin-yang relationship between the non-canonical inflammasome and MT3 in controlling inflammation and immunity to gram-negative bacteria.

Sex-Dependent Effects of Intestinal Microbiome Manipulation in a Mouse Model of Alzheimer's Disease.

Mechanisms linking intestinal bacteria and neurodegenerative diseases such as Alzheimer's disease (AD) are still unclear. We hypothesized that intestinal dysbiosis might potentiate AD, and manipulating the microbiome to promote intestinal eubiosis and immune homeostasis may improve AD-related brain changes. This study assessed sex differences in the effects of oral probiotic, antibiotics, and synbiotic treatments in the AppNL-G-F mouse model of AD. The fecal microbiome demonstrated significant correlations between bacterial genera in AppNL-G-F mice and Aß plaque load, gliosis, and memory performance. Female and not male AppNL-G-F mice fed probiotic but not synbiotic exhibited a decrease in Aß plaques, microgliosis, brain TNF-α, and memory improvement compared to no treatment controls. Although antibiotics treatment did not produce these multiple changes in brain cytokines, memory, or gliosis, it did decrease Aß plaque load and colon cytokines in AppNL-G-F males. The intestinal cytokine milieu and splenocyte phenotype of female but not male AppNL-G-F mice indicated a modest proinflammatory innate response following probiotic treatment compared to controls, with an adaptive response following antibiotics treatment in male AppNL-G-F mice. Overall, these results demonstrate the beneficial effects of probiotic only in AppNL-G-F females, with minimal benefits of antibiotics or synbiotic feeding in male or female mice.

Systems Genetics Approaches in Mouse Models of Group A Streptococcal Necrotizing Soft-Tissue Infections.

Mouse models are invaluable resources for studying the pathogenesis and preclinical evaluation of therapeutics and vaccines against many human pathogens. Infections caused by group A streptococcus (GAS, Streptococcus pyogenes) are heterogeneous ranging from mild pharyngitis to severe invasive necrotizing fasciitis, a subgroup of necrotizing soft-tissue infections (NSTIs). While several strains of mice including BALB/c, C3H/HeN, CBA/J, and C57BL/10 offered significant insights, the human specificity and the interindividual variations on susceptibility or resistance to GAS infections limit their ability to mirror responses as seen in humans. In this chapter, we discuss the advanced recombinant inbred (ARI) BXD mouse model that mimics the genetic diversity as seen in humans and underpins the feasibility to map multiple genes (genetic loci) modulating GAS NSTI. GAS produces a myriad of virulence factors, including superantigens (SAg). Superantigens are potent immune toxins that activate T cells by cross-linking T cell receptors with human leukocyte antigen class-II (HLA-II) molecules expressed on antigen-presenting cells. This leads to a pro-inflammatory cytokine storm and the subsequent multiple organ damage and shock. Inbred mice are innately refractive to SAg-mediated responses. In this chapter, we discuss the versatility of the HLA-II transgenic mouse model that allowed the biological validation of known genetic associations to GAS NSTI. The combined utility of ARI-BXD and HLA-II mice as complementary approaches that offer clinically translatable insights into pathomechanisms driven by complex traits and host genetic context and novel means to evaluate the in vivo efficiency of therapies to improve outcomes of GAS NSTI are also discussed.

Salivary Aß Secretion and Altered Oral Microbiome in Mouse Models of AD.

BACKGROUND: Beta amyloid (Aß) peptide containing plaque aggregations in the brain are a hallmark of Alzheimer's Disease (AD). However, Aß is produced by cell types outside of the brain suggesting that the peptide may serve a broad physiologic purpose. OBJECTIVE: Based upon our prior work documenting expression of amyloid ß precursor protein (APP) in intestinal epithelium we hypothesized that salivary epithelium might also express APP and be a source of Aß. METHODS: To begin testing this idea, we compared human age-matched control and AD salivary glands to C57BL/6 wild type, AppNL-G-F , and APP/PS1 mice. RESULTS: Both male and female AD, AppNL-G-F , and APP/PS1 glands demonstrated robust APP and Aß immunoreactivity. Female AppNL-G-F mice had significantly higher levels of pilocarpine stimulated Aß 1-42 compared to both wild type and APP/PS1 mice. No differences in male salivary Aß levels were detected. No significant differences in total pilocarpine stimulated saliva volumes were observed in any group. Both male and female AppNL-G-F but not APP/PS1 mice demonstrated significant differences in oral microbiome phylum and genus abundance compared to wild type mice. Male, but not female, APP/PS1 and AppNL-G-F mice had significantly thinner molar enamel compared to their wild type counterparts. CONCLUSION: These data support the idea that oral microbiome changes exist during AD in addition to changes in salivary Aß and oral health.

Double-Edged Role of Interleukin 17A in Streptococcus pneumoniae Pathogenesis During Influenza Virus Coinfection.

BACKGROUND: We sought to determine the role of host interleukin 17A (IL-17A) response against colonizing Streptococcus pneumoniae, and its transition to a pathogen during coinfection with an influenza virus, influenza A H1N1 A/Puerto Rico/8/1934 (PR8). METHOD: Wild-type (WT) C57BL/6 mice were intranasally inoculated with S. pneumoniae serotype 6A to establish colonization and later infected with the influenza strain, PR8, resulting in invasive S. pneumoniae disease. The role of the IL-17A response in colonization and coinfection was investigated in WT, RoRγt-/- and RAG1-/- mice with antibody-mediated depletion of IL-17A (WT) and CD90 cells (RAG1-/-). RESULTS: RAG1-/- mice did not clear colonization and IL-17A neutralization impaired 6A clearance in WT mice. RoRγt-/- mice also had reduced clearance. S. pneumoniae-PR8 coinfection elicited a robust IL-17A response in the nasopharynx; IL-17A neutralization reduced S. pneumoniae invasive disease. RoRγt-/- mice also had reduced S. pneumoniae disease in a coinfection model. Depletion of CD90+ cells suppressed the IL-17A response and reduced S. pneumoniae invasion in RAG1-/- mice. CONCLUSION: Our data show that although IL-17A reduces S. pneumoniae colonization, coinfection with influenza virus elicits a robust innate IL-17A response that promotes inflammation and S. pneumoniae disease in the nasopharynx.

Heterogeneity in FoxP3- and GARP/LAP-Expressing T Regulatory Cells in an HLA Class II Transgenic Murine Model of Necrotizing Soft Tissue Infections by Group A Streptococcus.

Invasive group A streptococcus (GAS) infections include necrotizing soft tissue infections (NSTI) and streptococcal toxic shock syndrome (STSS). We have previously shown that host HLA class II allelic variations determine the risk for necrotizing fasciitis (NF), a dominant subgroup of NSTI, and STSS by modulating responses to GAS superantigens (SAgs). SAgs are pivotal mediators of uncontrolled T-cell activation, triggering a proinflammatory cytokine storm in the host. FoxP3-expressing CD4+ CD25+ T regulatory cells (Tregs) comprise phenotypically and functionally heterogeneous subsets with a profound ability to suppress inflammatory responses. Specifically, activated Tregs, which express glycoprotein A repetitions predominant (GARP) and display latent transforming growth factor ß1 (TGF-ß1) complexes (latency-associated peptide [LAP]), exhibit strong immunosuppressive functions. The significance of Tregs that may participate in suppressing inflammatory responses during NSTI is unknown. Here, we phenotypically characterized FoxP3/GARP/LAP-expressing Tregs in GAS-infected or SAg (SmeZ)-stimulated splenocytes from transgenic (tg) mice expressing human HLA-II DRB1*15 (DR15 allele associated with nonsevere NF/STSS-protective responses) or DRB1*0402/DQB1*0302 (DR4/DQ8 alleles associated with neutral risk for combined NF/STSS). We demonstrated both in vivo and in vitro that the neutral-risk allele upregulates expression of CD4+ CD25+ activated effector T cells, with a significantly lower frequency of Foxp3+/GARP+ LAP- but higher frequency of Foxp3- LAP+ Tregs than seen with the protective allele. Additional in vitro studies revealed that the presentation of SmeZ by the neutral-risk allele significantly increases proliferation and expression of effector cytokines gamma interferon (IFN-γ) and interleukin-2 (IL-2) and upregulates CD4+ CD25+ T cell receptors (TCRs) carrying specific Vß 11 chain (TCRVß11+) T cells and Th1 transcription factor Tbx21 mRNA levels. Our data suggest that neutral-risk alleles may drive Th1 differentiation while attenuating the induction of Tregs associated with suppressive function.

Genetic Architecture of Group A Streptococcal Necrotizing Soft Tissue Infections in the Mouse.

Host genetic variations play an important role in several pathogenic diseases, and we have previously provided strong evidences that these genetic variations contribute significantly to differences in susceptibility and clinical outcomes of invasive Group A Streptococcus (GAS) infections, including sepsis and necrotizing soft tissue infections (NSTIs). Our initial studies with conventional mouse strains revealed that host genetic variations and sex differences play an important role in orchestrating the severity, susceptibility and outcomes of NSTIs. To understand the complex genetic architecture of NSTIs, we utilized an unbiased, forward systems genetics approach in an advanced recombinant inbred (ARI) panel of mouse strains (BXD). Through this approach, we uncovered interactions between host genetics, and other non-genetic cofactors including sex, age and body weight in determining susceptibility to NSTIs. We mapped three NSTIs-associated phenotypic traits (i.e., survival, percent weight change, and lesion size) to underlying host genetic variations by using the WebQTL tool, and identified four NSTIs-associated quantitative genetic loci (QTL) for survival on mouse chromosome (Chr) 2, for weight change on Chr 7, and for lesion size on Chr 6 and 18 respectively. These QTL harbor several polymorphic genes. Identification of multiple QTL highlighted the complexity of the host-pathogen interactions involved in NSTI pathogenesis. We then analyzed and rank-ordered host candidate genes in these QTL by using the QTLminer tool and then developed a list of 375 candidate genes on the basis of annotation data and biological relevance to NSTIs. Further differential expression analyses revealed 125 genes to be significantly differentially regulated in susceptible strains compared to their uninfected controls. Several of these genes are involved in innate immunity, inflammatory response, cell growth, development and proliferation, and apoptosis. Additional network analyses using ingenuity pathway analysis (IPA) of these 125 genes revealed interleukin-1 beta network as key network involved in modulating the differential susceptibility to GAS NSTIs.

Toxic shock syndrome: characterization of human immune responses to TSST-1 and evidence for sensitivity thresholds.

Noninvasive vaginal infections by Staphylococcus aureus strains producing the superantigen TSST-1 can cause menstrual toxic shock syndrome (mTSS). With the objective of exploring the basis for differential susceptibility to mTSS, the relative responsiveness to TSST-1 of healthy women has been investigated. Peripheral blood mononuclear cells from healthy donors were incubated with purified TSST-1 or with the T-cell mitogen phytohemmaglutinin (PHA), and proliferation was measured. The concentrations of TSST-1 and PHA required to elicit a response equivalent to 15% of the maximal achievable response (EC15) were determined. Although with PHA, EC15 values were comparable between donors, subjects could be classified as being of high, medium, or low sensitivity based on responsiveness to TSST-1. Sensitivity to TSST-1-induced proliferation was associated with increased production of the cytokines interleukin-2 and interferon-γ. When the entire T lymphocyte population was considered, there were no differences between sensitivity groups with respect to the frequency of cells known to be responsive to TSST-1 (those bearing CD3(+) Vß2(+)). However, there was an association between sensitivity to TSST-1 and certain HLA-class II haplotypes. Thus, the frequencies of DR7DQ2, DR14DQ5, DR4DQ8, and DR8DQ4 haplotypes were greater among those with high sensitivity, a finding confirmed by analysis of responses to immortalized homozygous B cell lines. Collectively, the results reveal that factors other than neutralizing antibody and the frequency of Vß2(+) T lymphocytes determine immunological responsiveness to TSST-1. Differential responsiveness of lymphocytes to TSST-1 may form the basis of interindividual variations in susceptibility to mTSS.

Staphylococcus aureus isolates encode variant staphylococcal enterotoxin B proteins that are diverse in superantigenicity and lethality.

Staphylococcus aureus produces superantigens (SAgs) that bind and cross-link T cells and APCs, leading to activation and proliferation of immune cells. SAgs bind to variable regions of the ß-chains of T cell receptors (Vß-TCRs), and each SAg binds a unique subset of Vß-TCRs. This binding leads to massive cytokine production and can result in toxic shock syndrome (TSS). The most abundantly produced staphylococcal SAgs and the most common causes of staphylococcal TSS are TSS toxin-1 (TSST-1), and staphylococcal enterotoxins B and C (SEB and SEC, respectively). There are several characterized variants of humans SECs, designated SEC1-4, but only one variant of SEB has been described. Sequencing the seb genes from over 20 S. aureus isolates show there are at least five different alleles of seb, encoding forms of SEB with predicted amino acid substitutions outside of the predicted immune-cell binding regions of the SAgs. Examination of purified, variant SEBs indicates that these amino acid substitutions cause differences in proliferation of rabbit splenocytes in vitro. Additionally, the SEBs varied in lethality in a rabbit model of TSS. The SEBs were diverse in their abilities to cause proliferation of human peripheral blood mononuclear cells, and differed in their activation of subsets of T cells. A soluble, high-affinity Vß-TCR, designed to neutralize the previously characterized variant of SEB (SEB1), was able to neutralize the variant SEBs, indicating that this high-affinity peptide may be useful in treating a variety of SEB-mediated illnesses.

Individual genetic variations directly effect polarization of cytokine responses to superantigens associated with streptococcal sepsis: implications for customized patient care.

Host immunogenetic variations strongly influence the severity of group A streptococcus sepsis by modulating responses to streptococcal superantigens (Strep-SAgs). Although HLA-II-DR15/DQ6 alleles strongly protect against severe sepsis, HLA-II-DR14/DR7/DQ5 alleles significantly increase the risk for toxic shock syndrome. We found that, regardless of individual variations in TCR-Vß repertoires, the presentation of Strep-SAgs by the protective HLA-II-DR15/DQ6 alleles significantly attenuated proliferative responses to Strep-SAgs, whereas their presentation by the high-risk alleles augmented it. Importantly, HLA-II variations differentially polarized cytokine responses to Strep-SAgs: the presentation of Strep-SAgs by HLA-II-DR15/DQ6 alleles elicited significantly higher ratios of anti-inflammatory cytokines (e.g., IL-10) to proinflammatory cytokines (e.g., IFN-γ) than did their presentation by the high-risk HLA-II alleles. Adding exogenous rIL-10 significantly attenuated responses to Strep-SAgs presented by the high-risk HLA-II alleles but did not completely block the response; instead, it reduced it to a level comparable to that seen when these superantigens were presented by the protective HLA-II alleles. Furthermore, adding neutralizing anti-IL-10 Abs augmented Strep-SAg responses in the presence of protective HLA-II alleles to the same level as (but no higher than) that seen when the superantigens were presented by the high-risk alleles. Our findings provide a molecular basis for the role of HLA-II allelic variations in modulating streptococcal sepsis outcomes and suggest the presence of an internal control mechanism that maintains superantigen responses within a defined range, which helps to eradicate the infection while attenuating pathological inflammatory responses that can inflict more harm than the infection itself.

In search of the identity of the XAP-1 antigen: a protein localized to cone outer segments.

PURPOSE: To determine the identity of the XAP-1 antigen. The XAP-1 antibody has been used by many investigators and is recognized as an index of photoreceptor outer segment maturity, yet its antigen remains unknown. METHODS: Previous studies documented that the XAP-1 antigen is a photoreceptor membrane-associated protein. To enrich for this protein, the authors prepared outer segment preparations from mouse retinas. Crude membrane and cytoplasmic fractions from this preparation were then generated using ultracentrifugation. Proteins were solubilized using n-dodecyl beta-D-maltoside and separated using SDS-PAGE. Aliquots of the crude membrane fraction were run on multiple lanes of a single gel, one lane of which was transferred to PVDF membrane and probed with the XAP-1 antibody. The remaining lanes were silver-stained. Very careful alignment of the Western blot with the silver-stained lanes indicated the presence of a single lightly stained band at the same position as the immunopositive band. nanoLC-ESI-MS/MS analysis was performed on the pooled protein bands. On determining the protein identity, confirmatory Western blot analysis and immunohistochemistry studies were performed. RESULTS: Western blot analysis performed using the XAP-1 antibody indicated a single immunoreactive band at approximately 74 kDa in lysates from both total outer segment and crude membrane preparations. No immunoreactive band was present in the cytoplasmic lysate. MS analysis of pooled silver stained bands determined that the XAP-1 antigen is Grp78. Western blot analysis and immunohistochemistry both support this identification. CONCLUSIONS: Present evidence indicates that the XAP-1 antigen is Grp78, a protein that has been previously documented in the interphotoreceptor matrix surrounding cones.

Proteomic analysis of the retina: removal of RPE alters outer segment assembly and retinal protein expression.

The mechanisms that regulate the complex physiological task of photoreceptor outer segment assembly remain an enigma. One limiting factor in revealing the mechanism(s) by which this process is modulated is that not all of the role players who participate in this process are known. The purpose of this study was to determine some of the retinal proteins that likely play a critical role in regulating photoreceptor outer segment assembly. To do so, we analyzed and compared the proteome map of tadpole Xenopus laevis retinal pigment epithelium (RPE)-supported retinas containing organized outer segments with that of RPE-deprived retinas containing disorganized outer segments. Solubilized proteins were labeled with CyDye fluors followed by multiplexed two-dimensional separation. The intensity of protein spots and comparison of proteome maps was performed using DeCyder software. Identification of differentially regulated proteins was determined using nanoLC-ESI-MS/MS analysis. We found a total of 27 protein spots, 21 of which were unique proteins, which were differentially expressed in retinas with disorganized outer segments. We predict that in the absence of the RPE, oxidative stress initiates an unfolded protein response. Subsequently, downregulation of several candidate Müller glial cell proteins may explain the inability of photoreceptors to properly fold their outer segment membranes. In this study, we have used identification and bioinformatics assessment of proteins that are differentially expressed in retinas with disorganized outer segments as a first step in determining probable key molecules involved in regulating photoreceptor outer segment assembly.

Aluminum hydroxide adjuvants activate caspase-1 and induce IL-1beta and IL-18 release.

Aluminum hydroxide (Alum) is the only adjuvant approved for routine use in humans, although the basis for its adjuvanticity remains poorly understood. In this study, we show that Alum activates caspase-1 and induce secretion of mature IL-1beta and IL-18. Human PBMC or dendritic cells stimulated with pure TLR4 and TLR2 agonists released only traces of IL-1beta or IL-18, despite the fact that the IL-1beta mRNA was readily induced by both TLR agonists. In contrast, cells costimulated with TLR agonists plus Alum released large amount of IL-1beta and IL-18. Alum-induced IL-1beta and IL-18 production was not due to enhancement of TLR signaling but rather reflected caspase-1 activation and in mouse dendritic cells occurred in a MyD88-independent fashion. Secretion of other proinflammatory cytokines such as IL-8 was not affected by Alum treatments. However, TLR-induced production of IL-10 was increased and that of IFN-gamma-inducible protein decreased by Alum cotreatment. Considering the immunostimulatory activities of these cytokines and the ability of IL-1beta to act as adjuvant, our results suggest a mechanism for the adjuvanticity of Alum.

Innate immune response to Francisella tularensis is mediated by TLR2 and caspase-1 activation.

Francisella tularensis, a gram-negative, facultative, intracellular bacterium, is the etiologic agent of tularemia and a category A bioterrorism agent. Little is known about the mechanism of pathogenesis of tularemia. In this paper, we describe the interaction of the live vaccine strain of F. tularensis with the innate immune system. We have found that in human and mouse dendritic cells, F. tularensis elicited a powerful inflammatory response, characterized by production of a number of cytokines and chemokines. Using cells derived from TLR2-deficient mice and in vitro transfection assays, we demonstrated that this response was mediated by TLR2 and did not require the LPS-binding protein. F. tularensis appeared to activate TLR2/TLR1 and TLR2/TLR6 heterodimers. IL-1beta secretion, a reflection of caspase-1 activation, was induced by live but not heat-killed F. tularensis, despite the fact that both forms of the bacterium equally induced the IL-1beta transcript. Our results identified activation of TLR2 and caspase-1 as the two main cellular pathways responsible for the inflammatory response to F. tularensis.

Impairment of tetanus-specific cellular and humoral responses following tetanus vaccination in human lymphatic filariasis.

To investigate the consequences of the impaired parasite-specific immune response in lymphatic filariasis, the effect of concurrent Wuchereria bancrofti infection on the immune response to tetanus toxoid (TT) following tetanus vaccination was studied in 20 asymptomatic microfilaremic (MF) patients, 20 patients with chronic lymphatic obstruction/elephantiasis (chronic pathology [CP]), and 10 endemic normal (EN) control individuals at baseline and at 3 and 6 months after TT vaccination. Peripheral blood mononuclear cell (PBMC) proliferative responses to TT before vaccination were not significantly different between the EN control and CP groups, but the MF group showed significantly lower baseline proliferative responses to TT compared with either the EN or CP group. Six months following vaccination, the change in proliferative response to TT was significantly greater in the EN and CP groups than in the MF group. This difference in proliferative response was reiterated in the gamma interferon (IFN-gamma) response in the EN group, in that they increased IFN-gamma production by 400% at 6 months, in contrast to that seen in the filaria-infected groups. In contrast to the IFN-gamma responses, PBMCs from the MF group produced significantly increased levels of TT-specific IL-10 compared with PBMCs from the EN group. Although there was significantly greater TT-specific immunoglobulin G (IgG) production at baseline between the EN and MF groups, postvaccination IgG (and IgG1 isotype) responses did not differ among the groups, whereas TT-specific IgG2, IgG3, and IgG4 were all increased in the EN group compared with the filaria-infected groups. These studies indicate that concurrent infection with W. bancrofti can diminish the immune response to an unrelated antigen by a mechanism that is likely to involve IL-10.