bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of a live attenuated bacterial vaccine for tularemia in the MIMIC® (Modular Immune In vitro Construct) System (52.6)
Authors: Moser Janice , Sassano Emily , Muerrle Christopher , Waag David , Wittman Vaughan
Published: January 2023

Sanofi Pasteur has optimized a series of unique, sensitive, automatable, and cost-effective assay systems, termed the Modular IMmune In vitro Construct (MIMIC®), to permit the evaluation of human innate and adaptive immunity in a laboratory setting. The lymphoid tissue equivalent (LTE) module of the MIMIC® platform reproduces the correct cellular interactions and kinetics to initiate primary (naïve) human B and T cell responses in an in vitro setting. We are currently investigating LVS, a live attenuated bacterial vaccine strain of Francisella tularensis, in the MIMIC® System. LVS generates a strong, naïve, TH1-skewed CD4 T cell response demonstrated by the percentage of responding cells producing intracellular IFNγ and TNFα. This data is consistent with human in vivo data showing that protection elicited by LVS vaccine includes a strong cellular immunity component. Previous studies have also shown that LVS drives production of protective antibodies in humans. We have currently demonstrated that the MIMIC® System can drive the generation of LVS-specific antibody responses in vitro using a fully autologous system. These studies indicate that the in vitro MIMIC® system offers a platform in which to conduct experiments in a fully human system prior to the involvement of human subjects. Therefore, the MIMIC® system provides a platform for analysis of new vaccine candidates and can be utilized for greater understanding of how efficient vaccines function.
Innate immune responses to polyanhydride nanoparticles modified with a toll like receptor three ligand. (52.9)
Authors: Huntimer Lucas , Carrillo-Conde Brenda , Petersen Latrisha , Ross Kathleen , Ramer-Tait Amanda , Bellaire Bryan , Narasimhan Balaji , Wannemuehler Michael
Published: January 2023

Improving efficacy of subunit-based immunization protocols without eliciting adverse reactions continues to be a hurdle in vaccine design. Vaccination of mice in our laboratory with surface eroding polyanhydride nanoparticles (NPs) prepared from 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) administered as a single intranasal dose provided high-titer and high-avidity humoral responses and protection against a live Yersinia pestis challenge at 23 weeks post-vaccination. The goal of the current study was to elucidate the immune-enhancing properties of the NPs by functionalizing them with TLR3 ligands (i.e., poly I:C) via either surface modification or encapsulation into the particle in order to activate macrophages. We also tested the hypothesis that the protective responses observed following intranasal NP vaccinations was due to particles undergoing endocytic processing by targeting TLR3 located on the endosomal membrane. Our results demonstrated that stimulation of primary macrophages (bone marrow-derived) with poly I:C modified particles increased surface expression of MHC II and CD80/86 as well as enhanced secretion of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α as compared to stimulation with non-functionalized NPs. Encapsulation of TLR ligands in polyanhydride NPs enhanced the activation status of macrophages and targeted functionalization may improve the efficacy of this novel vaccine platform.
Enhanced transgene expression improves immune responses following DNA vaccination (53.1)
Authors: Skinner Nicole , Dietz Wynette , Jund Michelle , Hwu Patrick , Chen Zhi-Ying , Kay Mark , Blazar Bruce , Pennell Christopher , Osborn Mark
Published: January 2023

Plasmid DNA (pDNA)-based gene delivery has the potential to advance vaccine development due to advantages such as low cost, ease of production, safety (e.g. pDNA does not cause insertional mutagenesis), and the flexibility to introduce several genes at once. To date, there are more than 300 clinical trials involving pDNA-based gene transfer. Although promising, these trials have revealed that poor transfection efficiency, transient protein production, and low immunogenicity limit therapy efficacy. We are investigating the use of minicircle (MC) DNA as a means of circumventing these limitations. MC vectors contain the expression cassette of full-length plasmids, but lack the bacterial backbone sequences that are targeted for transcriptional repression in mammalian cells. Removal of these plasmid backbone sequences is achieved via site-specific recombination in Escherichia coli prior to episomal (i.e. MC) DNA isolation. The absence of these prokaryotic elements also reduces MC size relative to its parental pDNA, leading to enhanced transfection efficiencies. Data will be presented showing that vaccination with MC DNA results in significantly higher and more prolonged reporter transgene expression. Furthermore, we compare specific responses of adoptively transferred and endogenous CD8+ T cells following vaccination of mice with either parental pDNA or MC-encoded antigen.
B cell immunotherapy to resolve a persistent viral infection (53.10)
Authors: McGavern Dorian , Heydari Sara
Published: January 2023

Many viruses can establish persistence and cause disease in humans. Studies have established a role for T cells in resolving persistent viral infections; however, the importance of a humoral response is less clear. Emerging evidence suggests that a coordinated effort between T and B cells is required to resolve some persistent infections. In this study, we sought novel insights into the importance of humoral immunity in resolving a persistent infection by utilizing the lymphocytic choriomeningitis virus (LCMV) model. Infection with aggressive LCMV variants results in a chronic viral infection during which anti-viral T cells pass through a state of functional exhaustion, but the infection is nevertheless controlled over time. We postulated that humoral immunity was required for LCMV control and could be harnessed as an immunotherapy. We observed plasma B cells in the repertoire as early as one week following LCMV infection, which coincided with the emergence of anti-viral antibodies (non-neutralizing). Depletion of B cells with anti-CD79 antibodies starting at the peak of T cell exhaustion (day 15) resulted in a failure of mice to control virus. Moreover, B cells harvested from mice that had controlled infection and transferred into mice with active infection resulted in accelerated viral control. These data demonstrate that humoral immunity can contribute to the resolution of a persistent viral infection and that B cells can be harnessed for adoptive immunotherapy.
Driving HIV-1 specific responses in immune suppressed recipients: Listeria as a vaccine vector (53.20)
Authors: McEwen Lisa , Bui Cac , Paterson Yvonne , Harn Donald
Published: January 2023

Malaria, TB and HIV-1 remain tremendous disease burdens in much of the world’s population and functional vaccines are desperately needed. Although Sub-Saharan populations are those that will benefit most from these vaccines, they are also coincident with areas of endemic helminth infection. Infection with one or more species of parasitic helminths suppresses the immune system and has been shown, by our lab and others, to suppress vaccine-specific responses. One goal of our research is to find vaccines that drive significant vaccine-specific immune responses in helminth infected recipients, without the need to eliminate helminth infection prior to vaccination. In the current study, we demonstrate that administration of a Listeria vector HIV-1 gag vaccine to mice chronically infected with the helminth parasite Schistosoma mansoni drives significant immune responses to HIV-1 gag CTL and helper epitopes. This observation suggests that Listeria vector vaccines are capable of driving vaccine-specific responses in helminth-infected populations. Kinetic studies show the antigen-specific responses are both durable and capable of inducing CD8+ central memory. In addition to HIV-1, Listeria vectors should be considered in the development of new generation malaria and TB vaccines to be administered to sub-Saharan Africa populations where helminth infection is endemic. Studies are underway to determine if other vectors are also capable of overcoming helminth-induced immune suppression.
Enhanced HIV-1 Gag-specific immunity induced by a sPD-1-based vaccine directed to dendritic cells (53.23)
Authors: Zhou Jingying , Wang Haibo , Tan Zhiwu , Du Yanhua , Chen Zhiwei
Published: January 2023

HIV/AIDS is a serious disease worldwide. The development of a safe, effective and affordable HIV-1 vaccine remains the ultimate solution. This study is based on the discovery of a negative immune regulatory system, the PD-1/PD-L pathway, which exists on both human and other animals. The PD-1 ligands (PD-L1 and PD-L2) are expressed on DCs. So we hypothesize to utilize the soluble PD-1 parts to intervene in the negative signal transmission of the PD-1 pathway and to target the antigen to DCs as a mean to indentify vaccine strategy to enhance the virus specific immune responses. Here we constructed the novel vaccine and controls by inserting soluble PD-1 gene and/or HIV-1 p24 gene into the pVAX1 vector with fusing of rabbit Fc fragment named mspd1-p24-fc, and controls named mspd1-IgVΔ-p24-fc and p24-fc. When compared with control vaccines, we discovered that mspd1-p24fc can significantly enhance HIV-1 Gag-specific immune responses by measuring the number of IFN-γ expressed CD4 and CD8 T cells using Elispot assays and CD8 T cell tetramer staining. Furthermore, the mspd1-p24-fc can significantly enhance humoral immune responses by anti-Gag antibody titer analysis. Importantly, this novel vaccine protects mice against recombinant vaccinia virus-gagpol challenges. This novel vaccine design may be used as DNA vaccine model against other infectious disease and cancer which need eliciting significant antigen specific humoral and cellular immune responses.
Impact of pre-existing immunity on the selection of rare adenovirus vector candidates: implications for HIV vaccine development (53.27)
Authors: Perreau Matthieu , Welles Hugh , Hall Olivia , Harari Alexandre , Bibert Stephanie , Martin Ricardo , Roger Thierry , Calandra Thierry , Bett Andrew , Gall Jason , Kremer Eric , Pantaleo Giuseppe
Published: January 2023

Adenovirus serotype 5 (Ad5) phase IIb vaccine trial (STEP) was prematurely stopped due to a lack of efficacy and two-fold higher incidence of HIV infection among Ad5 seropositive vaccine recipients. We have recently demonstrated that Ad5 immune complexes (Ad5 ICs)-mediated activation of the dendritic cell (DC)-T cell axis was associated with the enhancement of HIV infection in vitro. Although the direct role of Ad5 neutralizing antibodies (NAbs) in the increase of HIV susceptibility during the STEP trial is still under debate, vector-specific NAbs remain a major hurdle for vector-based gene therapies or vaccine strategies. To surmount this obstacle, vectors based on “rare” Ad serotypes including Ad6, Ad26, Ad36 and Ad41 were engineered. The present study aimed to determine whether Ad IC-mediated DC maturation could be circumvented using these Ad-vector candidates. We found that all Ad vectors tested forming ICs with plasma containing serotype-specific NAbs had the capacity to 1) mature human DCs as monitored by the up-regulation of co-stimulatory molecules and the release of pro-inflammatory cytokines (TNF-α), via the stabilization of Ad capsid at endosomal but not lysosomal pH rendering Ad DNA/TLR9 interactions possible and 2) potentiate Ad-specific CD4 and CD8 T cell responses. In conclusion, despite a conserved DC maturation potential, the low prevalence of serotype-specific NAbs renders rare Ad vectors attractive for vaccine strategies.
Human immune responses to Burkholderia pseudomallei characterized by protein microarray analysis (53.3)
Authors: Suwannasaen Duangchan , Mahawantung Jirawan , Chaowagul Wipada , Limmathurotsakul Direk , Felgner Philip L , Davies Huw , Bancroft Gregory J , Titball Richard W , Letmemongkolchai Ganjana
Published: January 2023

This study aimed to determine the antibody and T cell responses to Burkholderia pseudomallei (B. pseudomallei) of healthy individuals in endemic areas, and recovered melioidosis patients in order to select candidate vaccine antigens. A protein microarray of 154 (B. pseudomallei) proteins was probed with plasma from healthy individuals (n=108) and recovered patients (n=72). The results showed that 27 proteins distinctively reacted with plasma from both groups suggesting that different responses occur following environmental exposure or clinical melioidosis. We also compared the responses according to their history of subsequent relapse and the average antibody response to BPSL2765 (putative OmpA family protein) was over 10 times higher in plasma from individuals who had only one episode of disease than those with recurrent melioidosis. A comparison of antibody and T-cell responses to five (B. pseudomallei) proteins revealed that BimA and flagellin induced responses which were similar between both groups but BPSS0530; a conserved hypothetical protein could induce T cell responses of healthy controls more than recovered melioidosis. By combining large scale antibody microarrays and assays of T cell mediated immunity, we have identified a panel of novel (B. pseudomallei) proteins which show distinct patterns of reactivity in different stages of human melioidosis. These proteins may be useful candidates for development of subunit based vaccines.
TSG-6, a new protein ligand for CXCL8, modulates the activity of this pro-inflammatory chemokine (54.12)
Authors: Dyer Douglas , Milner Caroline , Day Anthony
Published: January 2023

TSG-6, a 35 kDa secreted protein, is expressed during inflammation (e.g. in the cartilage/synovium of arthritis patients) and delays the onset/reduces the severity of joint damage in models of inflammatory arthritis. For example, TSG-6-/- mice with proteoglycan-induced arthritis exhibit elevated neutrophil invasion and protease activity in the paw joints compared to controls. The aim of this study is to investigate potential mechanisms for the anti-inflammatory and chondroprotective effects of TSG-6, focusing on its interaction with CXCL8. This chemokine is known to promote neutrophil migration and to induce matrix metalloproteinase (MMP) secretion by endothelial cells. Using plate-based assays and surface plasmon resonance we have demonstrated that TSG-6 binds to CXCL8 in a pH-dependent manner and that TSG-6 can inhibit the interaction of CXCL8 with heparin. Since the sequestration of CXCL8 by endothelial glycosaminoglycans (e.g. heparan sulphate) contributes to its pro-migratory activity, this might represent one mechanism through which TSG-6 limits neutrophil extravasation. We have also shown, using gelatin zymography, that TSG-6 can block the CXCL8-mediated production of active MMPs by endothelial cells. MMPs have been implicated in the destruction of articular cartilage in arthritis, where they occur at elevated levels (e.g. in RA synovial fluids). Inhibition of the expression and/or activation of MMPs could, therefore, contribute to TSG-6’s chondroprotective effect.
Suppression of inflammation in collagen-induced arthritis by administration of recombinant Fcγ receptors (54.17)
Authors: Shashidharamurthy Rangaiah , Patel Jaina , Bozeman Erica , Selvaraj Periasamy
Published: January 2023

Recently we have shown that immune-complex mediated inflammation can be blocked by recombinant dimeric FcγR-Ig molecules in a mouse Arthus reaction model. In the present study, we have investigated whether administration of FcγR-Ig would inhibit the antibody-mediated hemolytic anemia, thrombocytopenia and arthritis in a mouse model. Systemic Administration of recombinant dimeric low affinity FcγRs such as human CD32A alleles (hCD32AR-Ig and hCD32AH-Ig) did not inhibit or prevent the antibody-induced hemolytic anemia and thrombocytopenia. Interestingly, we observed that after 48 hr of administration both hCD32AR-Ig and hCD32AH-Ig were able to inhibit 27% and 42% of the arthritic inflammation respectively, whereas the combination of hCD32AR-Ig and hCD32AH-Ig was able to inhibit 52% of arthritic inflammation. The severity of arthritis as assessed by inflammation and swelling in each digit and paw was significantly reduced. A single dose of hCD32A-Ig molecules was able to reduce the severity of arthritis up to 4 days, after which the disease returned and reached the maximum severity by day 7. The relapse of arthritis might be due to other mechanisms such as complement pathways or accelerated clearance of administered FcγR-Igs in arthritis mice. In conclusion, soluble dimeric FcγR-Igs could be of potential use in treating antibody-mediated autoimmune diseases such as arthritis but not for autoimmune diseases which affect blood cells such as hemolytic anemia and thrombocytopenia
In vivo regulation of IL-13 induced γδ T cell inflammatory responses by IL-17 (55.10)
Authors: Kinyanjui Margaret , Shan Jichuan , Fixman Elizabeth
Published: January 2023

The Th2 cytokine IL-13 regulates several aspects of asthma including airway inflammation, airway hyperresponsiveness (AHR) and mucus production. The Th17 cytokine, IL-17 is also known to participate in allergic disease since higher levels of IL-17 are associated with neutrophilia, particularly in severe or fatal asthma. To better understand the interaction of these cytokines in vivo, we intranasally administered recombinant IL-13 and/or IL-17 to anaesthetized mice. Administration of IL-13 alone led to an increase in airway inflammation and AHR. Intracellular cytokine staining of the recovered bronchoalveolar lavage cells revealed increased numbers of IL-17 producing T cells (both γδ and CD4). Administration of IL-17 alone had no effect on these experimental outcomes but did increase levels of tyrosine phosphorylated STAT3 in the lungs compared to saline controls. Treatment with both IL-13 and IL-17 led to an increase in the number of infiltrating eosinophils. While co-administration of IL-17 (with IL-13) had no effect on the number of infiltrating IL-17+ve CD4 cells, it decreased the number of IL-17+ve γδ T cells in the bronchoalveolar lavage. IL-17 also inhibited IL-13-induced increases in mRNA encoding IL-17, TGF beta and IL-6. Further studies examining the mechanism by which γδ T cells and IL-17 associated mediators are regulated by IL-13 and IL-17 are under way.
The effect of bee venom on ovalbumin-induced asthma model (55.13)
Authors: Park Soojin , Kim Jinju , Shin Minkyu , Hong Moochang , Bae Hyunsu
Published: January 2023

Asthma is a chronic disease marked by airway hyperreactivity and inflammation. Bee venom(BV) has been traditionally used in oriental medicine to relieve pain and to treat chronic inflammatory diseases. Some recent reports suggest that BV maybe an effective treatment for patients with asthma. However, there are still some unanswered questions relating to efficacy of BV therapy in asthma. The CD4+CD25+Foxp3+ regulatory T cell(Treg) play an important role in regulating the immune response. Treg can reduce inflammation and allergic reactions through its inhibitory functions. In this study, we reasoned that BV inhibits inflammatory diseases by effect on Treg and examined the effect of BV in naïve mice. Diverse concentration was treated everyday for 5days and BV significantly expanded Treg on 1mg/kg concentration. This result proposes that BV can attenuate asthma. We used 7-10 weeks female Balb/c mice were sensitized with ovalbumin(OVA). As a result, BV decreased inflammatory cytokine secretion including IL-4, IL-5, IL-13 in bronchoalveolar lavage(BAL) fluid. Also BV was significantly decrease structural remodeling of the airway. The number of Neutrophil, Lymphocyte, Macrophage, and total cells were significantly lower in the BV group than the OVA group. These results suggest that BV has potential therapeutic value for controlling asthma responses resulting from exposure to allergens.
Fullerene C70 derivatives inhibit mast cell mediated airway inflammation and bronchoconstriction associated with allergic asthma (55.2)
Authors: Norton Sarah , Dellinger Anthony , Zhou Zhiguo , Conrad Daniel , Kepley Christopher
Published: January 2023

Fullerenes are carbon nanospheres that can be solublized by the addition of polar chemical groups to the carbon cage. Tetragylocolate (TGA) is a derivatized fullerene compound that has been shown to stabilize mast cell (MC) responses in vitro (Clin Trans Sci. Vol: 1-11), thus we hypothesized it may have an effect on MC-driven diseases such as asthma. To observe the effects of TGA on chronic features of asthma in vivo, we utilized a MC-dependent model of asthma pathogenesis. Mice were sensitized by intraperitoneal injection of ovalbumin (OVA) in saline, challenged intranasally (i.n.) with OVA, and one of two treatment strategies was pursued. In one, TGA was given i.n. throughout disease development. In the other, TGA was given following an initial set of challenges to allow disease to develop prior to treatment; mice were then re-challenged with OVA to assess the effect on established disease. Mice did not exhibit any acute toxicity and liver and kidney function were normal. We find that TGA treatment significantly reduces airway inflammation and eosinophilia and dramatically reduces bronchoconstriction in either model. This may be due in part to significant reductions in IL-4 and IL-5 levels in the lungs. Studies utilizing fluorescently labeled TGA show this compound localized within mast cells in murine lung tissue, suggesting in vivo mast cell inhibition. Studies are in progress to determine in vivo effects of TGA on mast cell expression of asthma related genes.
Immunocomplexes down-modulate the induction of food allergy in mouse through CD32b- and IL-21-dependent mechanisms. (55.7)
Authors: Araujo Claudia , Ganeshan Kirthana , Bryce Paul , Sperling Anne
Published: January 2023

Food allergy is an adverse Th2 response whose incidence has been increasing dramatically. Previously, we have demonstrated that immunocomplexes(IC) decrease the food allergic responses. Thus, we hypothesize that inhibitory FcR is triggered by IC in food allergy. Here we investigated the contribution of CD32b signaling in the down-modulatory effect of IC in a mouse food allergy model. Wild type(WT) or CD16-/- or CD64-/- or CD32b-/- or IL-21R-/-mice were sensitized by gavage for 8 weeks with SEB, SEB+OVA or SEB+OVA-IC and challenged on the 9th week with OVA. We demonstrated that OVA-IC-sensitized WT, CD16-/- or CD64-/- mice produced lower levels of serum IgE, IL-4, IL-5, and have decreased blood eosinophilia compared to OVA group. In contrast, OVA-IC-sensitized CD32b-/- mice did present food allergic responses similar to OVA group. In addition, IL-21 levels were high in OVA-IC-sensitized WT, CD16-/-, CD64-/- mice, but in CD32-/- mice, in comparison to OVA group. Besides, OVA-IC-sensitized IL-21R-/- mice restored the food allergic responses, similar to OVA group. In conclusion, IC down-regulates the food allergic response in WT, CD16-/-, CD64-/- mice but in CD32b-/- indicating that CD32b plays a role in the down-modulatory effect of IC. Besides, IC stimulate the production of IL-21 and up-regulate food allergic responses in IL-21R-/- mice, suggesting that down-modulation induced by IC is mediated by IL-21-dependent mechanism.
Absence of the Type I IL-4 Receptor reduces IFNγ production and increases the severity of airway inflammation in an ovalbumin induced model of asthma (55.8)
Authors: Dasgupta Preeta , Smith Elizabeth , Keegan Achsah
Published: January 2023

The TH2 cytokines, IL-4 and IL-13, play critical roles in inducing allergic lung inflammation and also drive alternative activation of macrophages (AAM). Although both cytokines share receptor subunits and signaling proteins, there is evidence that IL-4 and IL-13 have differential roles in asthma pathogenesis. While important in TH2 differentiation, the contribution of IL-4 signaling via the Type I receptor, in modulating airway inflammation remains unclear. Here, we adoptively transferred wild-type ova primed CD4+ T cells into C57BL/6, Rag2KO, or gamma c (γc) KO mice. γcKO mice developed increased peribronchial and perivascular inflammation in the lungs upon ova challenge, compared to WT and Rag2KO mice. Moreover, significantly higher numbers of eosinophils were present in the bronchoalveolar lavage (BAL) and lung tissue in γcKO mice. Characteristic AAM proteins FIZZ1 and YM1 were expressed in lung epithelial cells in both mouse strains, but greater intensity of YM1 expression was observed in γcKOs. The number of FIZZ1+ or YM1+ airways was also significantly higher in these mice. It is known that γcKO mice lack NK cells; Type I R signaling in NK cells induces IFNγ production. Indeed, we found reduced secretion of IFNγ in the BAL in γcKO mice as compared to Rag2KO mice. These results suggest that the Type I R acts to suppress allergic lung inflammation through IFNγ production and, in the presence of TH2 effectors, the Type II R can mediate allergic responses in its absence.
Inhibitory effects of 6-(phenylsulfonyl)nicotinonitrile against Vibrio vulnificus infection to host (56.16)
Authors: Kim Myun Soo , Lim Dong Yeol , Choi Sang Ho , Kim Tae Sung
Published: January 2023

Vibrio vulnificus is a gram-negative bacterium that is present in marine environment, and infection of the bacteria shows rapid pathological progress. V. vulnificus induces the fever, septic shock, the formation of secondary lesions, accompanied in many cases by ulcer and edema on the patients. Mortality of primary septicemia exceeds 50 % in septic patients. In this study, we examined the antimicrobial activity of 6-(phenylsulfonyl)nicotinonitrile, named U262, against V. vulnificus in vitro and in vivo. Treatment with U262 significantly inhibited the V. vulnificus-induced cytotoxicity in human intestinal epithelial INT-407 cells. U262 itself was not toxic to host cells, and did not affect the growth of the bacteria. Interestingly, U262 treatment significantly decreased the attachment of V. vulnificus to the INT-407 cells. Furthermore, the administration of U262 increased the survival period of V. vulnificus-infected mice. The numbers of viable V. vulnificus in the small intestine, liver, spleen and blood were significantly lower in the U262-treated mice, compared with those of the untreated mice. Taken together, these results demonstrate that U262 suppresses the V. vulnificus-mediated cytotoxicity in human intestinal epithelial cells, maybe via the down-regulation of bacterial attachment to the epithelial cells, and suggest that U262 can be utilized as a novel candidate in the treatment of V. vulnificus infection.
Lung macrophages control malaria-induced pulmonary inflammation (56.17)
Authors: Lagasse H.A. Daniel , Scott Alan
Published: January 2023

Pulmonary edema and acute respiratory distress are archetypal symptoms of severe malaria that occur in ~20% of patients and are associated with mortality rates exceeding 70%. Little is known about the pathogenesis of lung pathology associated with Plasmodium infections. We tested the hypothesis that two major cellular populations - CD11c+CD11b- resident macrophages and CD11b+Ly6C+ recruited monocytes/macrophages - are key players in regulating the nature and magnitude of malaria-induced pulmonary pathology. During the very early stages of Plasmodium berghei infections in mice, parasites are taken up by resident macrophages. This is associated with the transient activation of resident macrophages and the release of chemokines that recruit large numbers of CD11b+Ly6C+ blood monocytes. Flow cytometry results suggest that later in infection resident lung macrophages take on a regulatory phenotype, while the recruited monocytes undergo activation, differentiation into macrophages and are responsible for the majority of parasite clearance within the lungs. Administration of a blocking monoclonal antibody (anti-CD11b; 5C6) diminishes the homing of monocytes to the lungs, resulting in decreased parasite uptake. We propose that both myeloid populations control pulmonary inflammation through the clearance of sequestered parasites within the pulmonary microvasculature as well as the regulation of pro-inflammatory processes within the lung environment.
Borrelia burgdorferi, the spirochetal agent of Lyme disease, induces type I interferon by human immune cells via detection of DNA and TLR7-dependent recognition of RNA. (56.19)
Authors: Love Andrea , Schwartz Ira , Petzke Mary
Published: January 2023

Borrelia burgdorferi (Bb) is the spirochetal agent of Lyme disease, the most common tick-transmitted infection in the United States. This extracellular pathogen initiates a type I interferon (IFN) response following phagocytosis and recognition of live Bb by toll-like receptors (TLRs) 7 and 9. To identify the ligands of Bb that induce the type I IFN response in human cells, cellular components of Bb were delivered into peripheral blood mononuclear cells (PBMCs) by encapsulation with DOTAP. Type I IFN-responsive genes (MX1, OAS1, IRF7) were significantly induced by RNA, DNA, and cell lysates relative to unstimulated PBMCs (p<0.05 for all). Unencapsulated lysate or lysate treated with both RNase and DNase were unable to elicit this response. In contrast, proteinase K treatment of lysate had no effect. Purified, DOTAP-encapsulated Bb DNA and RNA induced production of significant levels of IFN-α (p<0.01). The IFN response to RNA was abolished with RNase treatment or by specific inhibition of TLR7 (p<0.01). This demonstrates that Bb RNA is the ligand for TLR7 in human cells, contributing to IFN-α production and induction of IFN-responsive genes. These results support prior data demonstrating that the type I IFN response requires phagocytosis and is not mediated by a cell surface receptor in human cells. Taken together, these data demonstrate that RNA (via TLR7) and DNA, but not protein, are the Bb cellular components that initiate the type I IFN response in human PBMCs.
IL-10 elicited during malaria infection blunts effective immune responses to non-typhoidal Salmonella (56.32)
Authors: Mooney Jason , Butler Brian , Xavier Mariana , Chau Jennifer , George Michael , Dandekar Satya , Luckhart Shirley , Tsolis Renee
Published: January 2023

Co-infection of non-typhoidal Salmonella serotypes (NTS) in pediatric patients with severe Plasmodium falciparum malaria can develop a life threatening bacteremia, a major source of child mortality in Africa. Here, we use a mouse model that mimics severe anemia seen in human malaria to address mechanisms by which an underlying malaria parasite infection contributes to the increased risk of developing NTS bacteremia. Plasmodium yoelli infected red blood cells were administered intraperitoneally in CBA, B6 or B6 il10-/- mice. At peak parasitemia, Salmonella Typhimurium was administered by gavage in a streptomycin-induced colitis model. Characterization of inflammation to NTS in the cecum was assessed by histopathology, microarray and qRT-PCR. Bacterial loads in systemic tissues were followed to 4 days post infection. Our results indicate that the malaria parasite infection causes a global suppression of proinflammatory responses in the intestine, blunts the intestinal neutrophil influx normally elicited during NTS gastroenteritis and increases bacterial colonization at systemic sites. Blunting of intestinal inflammatory responses was independent of hemolytic anemia, but required induction of the immunoregulatory cytokine IL-10 by the parasites. Elimination of IL-10 activity restored intestinal inflammation in co-infected mice, and administration of recombinant IL-10 was sufficient to increase systemic colonization by S. Typhimurium in the absence of parasite infection.
Chitinase activity and a novel population of CXCR3 expressing alternatively activated macrophages in the CNS during chronic Toxoplasma gondii infection. (56.33)
Authors: Nance J. , Noor Shahani , Lane Thomas , Wilson Emma
Published: January 2023

During infection with Toxoplasma gondii, an IFN-γ dependent polarization of classically activated macrophages (CaM) is required to control parasite growth during the acute phase of infection. These macrophages produce high levels of cytokines such as IL-12 and TNF-α and other effector molecules such as nitric oxide that act to induce stage conversion to bradyzoites resulting in a sustained chronic infection in the brain. Cytokines such as IL-4 as well as the presence of exogenous polymers such as chitin, induce alternatively activated macrophage (AMac) phenotypes involved in tissue remodeling and wound healing responses. Here we demonstrate the presence of AMac macrophages during Toxoplasma infection at a time point associated with chronic infection and substantial tissue remodeling in the brain. This population preferentially expressed the chemokine receptor CXCR3, scavenger receptors, MMR and stabilin-1. In vivo blockade of CXCR3 and CXCL10 decreased not only T cell infiltration and resulted in an increase in parasite burden but macrophage migration to the brain was also diminished. Finally, we demonstrate chitinase activity in the Toxoplasma infected brain associated with the presence of AMac and chitin in the cyst wall. These results indicate that chitin in the parasitic cyst wall locally stimulates a population of CXCR3 expressing AMac that are required for tissue remodeling and immunity in the brain during Toxoplasma infection and inflammation.
IL-1 receptor antagonist augments antibiotic resolution of joint inflammation in a novel model of Brucella-induced osteoarthritis (56.7)
Authors: Skyberg Jerod , Thornburg Theresa , Kochetkova Irina , Layton William , Callis Gayle , Riccardi Carol , Golden Sarah , Rollins MaryClare , Pascual David
Published: January 2023

Infection of the joints is the most frequent localized manifestation of brucellosis, which is a common cause of infectious arthritis. However, no experimental murine model of Brucella-induced arthritis has been reported. Here we report that IFN-γ-/- mice develop joint inflammation following oral, nasal, or parenteral infection with B. abortus or B. melitensis. Joints from Brucella-infected IFN-γ-/-, but not wild-type mice, were found to contain extensive inflammatory infiltrates and debris within the joint space which co-localized with brucellae. Osteoarthritis, joint space narrowing, necrosis, soft tissue inflammation, and substantial Brucella burdens were also observed, although antibody or cytokine responses against collagen were not detected. Elevated IL-1β, but not TNF-α, IL-6, nor IL-17, was detected in the joints of Brucella-infected IFN-γ-/- mice. A six-week regimen of rifampicin effectively cleared infection and halted further progression of inflammation, although some symptoms and swelling remained. However, administration of an adenovirus expressing the IL-1receptor antagonist augmented antibiotic resolution of joint swelling by >50%. These results show that the IFN-γ-/- mouse represents a useful model to study the pathogenesis of joint inflammation due to Brucella infection, and that intervention strategies targeting IL-1 can complement antibiotic treatment of Brucella-induced inflammation. Supported by USDA 2007-01612 and USDA 2009-34397-20133.
IL-10 from macrophages and granulocytes is not required for chronic disease in Leishmania mexicana infection (56.8)
Authors: Buxbaum Laurence
Published: January 2023

Chronic disease of mice caused by the protozoan parasite Leishmania mexicana requires IL-10 and FcγRIII. Immune complexes consisting of IgG bound to the surface of Leishmania amastigotes can induce IL-10 and suppress IL-12 production from macrophages in vitro. We have found that IgG1 and IgG2a/c can each efficiently induce IL-10 from macrophages, whereas in vivo, mice that lack IgG1 and have stronger and earlier IgG2a/c responses, are more resistant to L. mexicana infection. This calls into question the crucial role of macrophages as the source of IL-10 required for chronic disease. We now show that tissue-specific IL-10-deficient mice lacking IL-10 from macrophages and granulocytes still have chronic disease similar to WT C57BL/6 mice. These Lyz2-cre-IL-10fl/fl mice clearly lack IL-10 from macrophages as expected, but have a similar course of infection and parasite loads as the WT mice. This shows more directly that macrophage IL-10 is not required for chronic disease and that neutrophils are also not a required source of IL-10. Prior work showed that depletion of CD25+CD4+ T cells also produced no major change in chronic disease and that T cell IL-10 in lymph nodes did not differ in mice that heal (such as FcγRIII KO mice) and those that have chronic disease (WT mice). This leaves an open question as to the important source of IL-10, likely in lesions rather than in the lymph node.
Soluble IL7 receptor alpha chain serves as a depot to prevent IL7 consumption (57.1)
Authors: Lundstrom Wangko , Walsh Scott , Mackall Crystal
Published: January 2023

IL7 signals through a receptor comprised of IL7Ra and IL2RG. Association studies have shown IL7R genotype modulates susceptibility to MS. The MS associated allele also increases an alternatively spliced mRNA isoform encoding a soluble receptor (sIL7Ra). Thus sIL7Ra seems to be associated with increased MS risk, however the biological mechanism behind this is poorly understood. We want to determine the effect of sIL7Ra on IL7 signaling as a first step toward determining how polymorphisms in IL7R modulate risk for MS.We produced recombinant isoform encoded sIL7Ra and recombinant extracellular IL7Ra (i.e. shed IL7Ra) and measured binding to IL7 using Biacore. Both proteins show intermediate affinity binding to IL7, with sIL7Ra demonstrating 15-fold increased affinity (Kd=6.3nM) compared to extracellular IL7Ra (Kd=98nM). We measured the effect of sIL7Ra on IL7 mediated cell survival using 2E8, an IL7 dependent cell line. At day 3 sIL7Ra diminished IL7 mediated survival, however at day 9 and 12 the effect is reversed and presence of sIL7Ra increases survival compared to cells cultured with IL7 alone.In conclusion, sIL7Ra competes with membrane bound IL7Ra for IL7 binding. This initially decreases IL7 signaling, but later on IL7 is preserved in the presence of soluble receptor. We postulate that sIL7Ra serves as a depot for IL7, resulting in more persistent IL7 signal over time. Increased levels of soluble IL7Ra may increase the risk of MS by maintaining stronger IL7 signals.
The essential role of IL-7 signalling in CD8+ T-cell activity (57.17)
Authors: Crawley Angela , Agatha Vranjkovic , McGuinty Mikki , Faller Elliott , MacPherson Paul , Angel Jonathan
Published: January 2023

Anti-viral CD8+ T-cell responses become impaired in HIV infection in part due to decreased T-cell survival, function and memory cell development; aspects mediated largely by interleukin-7 (IL-7). In progressive HIV infection, decreased T-cell expression of the IL-7 receptor α (CD127) and impaired IL-7 signalling, despite increased IL-7 production, may contribute to failing T-cell activity. Here we examine the effects of IL-7 on a panel of signalling pathways and investigates how IL-7 signalling pathways are associated with IL-7-related activities in CD8+ T-cells. Low concentrations of IL-7 (10 pg/ml) are capable of inducing maximum activation of the Jak-STAT and PI3K signalling pathways, while higher concentrations (500-1000 pg/ml) were required to induce Bcl-2 production and glucose uptake. Even higher concentrations of IL-7 (10,000 pg/ml) were needed to induce cell proliferation and perforin release. Inhibition of Jak activation reduced IL-7-induced Bcl-2, perforin production and proliferation. The activation of intracellular signalling pathways by IL-7 in human CD8+ T-cells and how they are associated with IL-7-induced functions has been extensively studied. Furthermore, the kinetic and optimal concentrations of IL-7 required for the induction of these functional outcomes suggest a complex control of IL-7-associated functions. This may provide insight in immune restoration therapies in HIV infection, where IL-7 signalling and functions are impaired.
Detection of a point mutation in IL12/23 receptor beta 1 chain in a Mexican patient with uncommon clinical feature. (58.5)
Authors: Ramirez Alejo Noe , Estrada Garcia Iris , Blancas Galicia Lizbeth , Espinosa Rosales Francisco , Santos Argumedo Leopoldo
Published: January 2023

Mendelian susceptibility to mycobacterial disease (MSMD) is a heterogenous syndrome. The clinical characteristics include repetitive local infection and in some cases dissemination. In this work we analyzed a 5 years old boy with an uncharacterized mycobacterial infection. His parents are first degree cousins. Blood samples were collected and cultured 48 hours with medium, BCG or BCG+IL-12. Supernatants were assayed for IFN-γ by ELISA. PBMC were cultured with PMA-Ionomicyn and IL12R was measured by flow cytometry. We found low production of IFN γ after stimulation with BCG+IL12 that seems to correlate with an IL-12R defect. Flow cytometry showed a decrease of IL-12RB1 expression. Sequencing revealed a point mutation (A182T), which generates E61G change in the extracellular domain. Furthermore, the uncommon clinical features in this case might suggest another primary immunodeficiency.
Augmented Th2 response and liver fibrosis in mice lacking the chitinase like protein, BRP-39 (58.9)
Authors: Ramalingam Thirumalai , Barron Luke , Mentink-Kane Margaret , Cheever Allen , Humbles Alison , Kolbeck Roland , Wynn Thomas
Published: January 2023

The mouse Breast Regression Protein 39 (BRP39; Chi3l1) is a chitinase like protein but lacks enzymatic activity. Recently it was shown that mice lacking BRP39 displayed significantly impaired Th2 response during allergic airway disease. We sought to examine if the absence of BRP39 would affect the generation of protective and pathogenic Th2 responses following helminth infections. In a model of Schistosoma mansoni egg induced pulmonary granuloma formation, naive BRP39KO animals developed weaker Th2 response upon egg embolization. The Th2 responses were restored to WT levels if pulmonary granulomas were elicited in antigen primed (secondary granuloma) animals. However, the size of secondary egg granulomas remained significantly smaller in the KO animals, suggesting a role for BRP39 in regulation of pulmonary granuloma response. In contrast, following infection with S. mansoni cercariae, BRP39KO animals manifested larger hepatic granulomas and significantly exacerbated liver fibrosis and showed an increased prevalence of CD4+ cells making IL-5 and IL-13 among the granuloma leukocytes. Furthermore, Nippostrongylus brasiliensis infected BRP39KO mice displayed no defect in worm expulsion suggesting normal Th2 response in the gut. Thus, these studies reveal that the defectiveTh2 response in the absence of BRP39 is a transient phenomenon and introduce the possibility that BRP39 could negatively regulate Th2 responses in a chronic setting and in a tissue specific manner.
Transgenic human IL-10 expression by CD4+ T cells is associated with resistance to colitis in Il10-/- mice (59.11)
Authors: Bream Jay , Ramakrishnan Amritha , Wang Fengying , Brayton Cory , Ranatunga Dilini
Published: January 2023

Studies in mice suggest that the cellular source of the immunoregulatory cytokine IL-10 plays a key role in determining susceptibility to various infectious and autoimmune diseases. To better understand cell-specific human IL-10 (hIL-10) expression we generated a hIL-10 transgenic mouse using a bacterial artificial chromosome (hIL10BAC). Human IL-10 is biologically active in the mouse, and by reconstituting Il10-/- mice with the hIL10BAC we can evaluate the effects of human IL-10 regulation in vivo. We found that the hIL10BAC rescues Il10-/- mice from helicobacter-induced colitis as evidenced by histopathology scores of inflammation and a failure to develop rectal prolaspe. Aberrant proinflammatory cytokine mRNA expression in the colons of Il10-/- mice was reduced in Il10-/-/hIL10BAC mice. Likewise, the percentages of IL-17-expressing CD4+ T cells in the ileal and colonic lamina propria were normalized to WT levels in Il10-/-/hIL10BAC mice. In searching for cellular sources of hIL-10 we found that hIL-10 expression was enriched specifically within the colonic lamina propria and these cells were CD4+CD25+Foxp3+ T cells. Adoptive transfer of CD4+ Il10-/-/hIL10BAC T cells protected Rag1-/- mice from wasting disease using a T cell transfer model of colitis. These data combined with previous results from our laboratory suggests that human IL-10 expression in various CD4+ T cell subsets is under different regulatory constraints.
Transfer of IgG in the female genital tract by the FcRn confers protective immunity to vaginal infection (59.3)
Authors: Zhu Xiaoping , Li Zili , Palaniyandi Senthilkumar , Zeng Rongyu , Tuo Wenbin , Roopenian Derry
Published: January 2023

IgG is a major Ig subclass in mucosal secretions of the human female genital tract, where it predominates over the IgA isotype. Despite the abundance of IgG, surprisingly little is known about where and how IgG enters the lumen of the genital tract and the exact role local IgG plays in preventing sexually transmitted diseases. We demonstrate here that the FcRn is expressed in female genital tract epithelial cells of humans and mice and binds IgG in a pH dependent manner. In vitro we show that FcRn mediates bidirectional IgG transport across polarized human endometrial HEC-1-A monolayers and primary human genital epithelial cells. Furthermore, endosomal acidification appears to be a prerequisite for FcRn-mediated IgG transcytosis; IgG transcytosis was demonstrated in vivo by translocation of systemically administered IgG into the genital lumen in WT but not FcRn-KO mice. The biological relevance of FcRn-transported IgG was demonstrated by passive immunization using HSV-2-specific polyclonal serum, which conferred significantly higher protection against intravaginal challenge infection by the HSV-2 186 strain in WT mice than in FcRn-KO mice. These studies demonstrate that FcRn-mediated transport is a mechanism by which IgG can act locally in the female genital tract in immune surveillance and in host defense against sexually transmitted diseases.
Macrophage migration inhibitory factor promotes central nervous system pathology through local microglial activation and macrophage trafficking (60.3)
Authors: Cox Gina Mavrikis , Alexander J. , Kithcart A. , Williams J. , Smith K. , Shawler T. , Guan Z. , Satoskar A. , Popovich P. , Whitacre C.
Published: January 2023

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). It is reported that cerebrospinal fluid from MS patients during relapse contains higher levels of MIF than samples collected during remission. While the potent pro-inflammatory properties of MIF are well established, the precise contribution to neuroinflammation is not well defined. We have previously shown that despite their ability to generate pathogenic T cells, MIF-/- mice fail to develop experimental autoimmune encephalomyelitis (EAE), and have reduced CNS pathology. These findings suggest that MIF acts within the CNS to potentiate disease progression. We observed that MIF-treated microglia upregulate inflammatory mediators associated with MS and EAE including IL-1, IL-6, TNF-α, iNOS, and CCL2, and exhibit morphological changes consistent with activation. We extended these findings in vivo, performing stereotactic spinal microinjection of rMIF into naïve mice, which resulted in targeted microglial reactivity and cellular infiltration. To determine the role of MIF in the CNS during EAE, we examined the ability of rMIF spinal microinjection to restore CNS pathology in MIF-/- mice. Notably, rMIF microinjection was sufficient to restore CNS pathology in EAE-resistant MIF-/- mice following induction of EAE. Taken together, these studies implicate MIF as a CNS inflammatory mediator that may contribute to the development and maintenance of MS lesions.