bluuebunny/crossref_metadata_2025 huggingface dataset:
The multiplication of invasive Streptococcus pyogenes in endothelial cell accompanies autophagy formation (40.13)
Authors: Lu Shiou-Ling , Lin Yee-Shin
Published: January 2023

Streptococcus pyogenes, also known as group A streptococcus (GAS), is one of most common human pathogens. Severe life-threatening disease, such as sepsis, results from the breakdown of blood vessel barrier. Although GAS has been identified as an extracellular bacterium, internalization of GAS by many eukaryotic cells may provide a mechanism of clinical severe damage due to evading from immune surveillance or antibiotic treatment. Previous report showed that GAS can be efficiently killed by autophagy (an intracellular innate immune system) in some nonphagocytes including epithelial cells. In this study, we showed that invasive GAS, but not noninvasive GAS strains, multiplied inside endothelial cells and bacterial growth reached plateau at 6 ~ 8 h. The intracellular sites of multiplication of GAS were first in vesicles and then in cytoplasm where most of the GAS was liberated from vesicle by streptolysin before endosomal degradation. GAS further induced autophagy formation but only a small portion of GAS was completely enveloped by GAS-containing autophagosome-like vacuoles at 6 h. Furthermore, endothelial cells underwent a caspase-indepandent cell death caused by persistent multiplication of GAS. The interrelationship between autophagy induction, cell death, and GAS multiplication remains further investigation. Our studies demonstrate that the insufficient autophagy induction during invasive GAS infection, particularly in endothelial cells, results in multiplication of GAS.
T lymphocytes are essential for initiation of fungal keratitis in mice (40.14)
Authors: Wang Yiqiang , Zhang Hongbo , Jia Changkai , Xi Haijie , Li Siyuan
Published: January 2023

In preliminary study, we found that experimental fungal keratitis (FK) induced an adaptive immune response that involved T lymphocytes infiltration and antibody accumulation in corneas. Here we explored the role of T lymphocytes in the progess of FK. Immunocompetent Balb/c mice and nude mice on Balb/c background were utilized. For adoptive transfer of lymphocytes, B cells and T cells were isolated from Balb/c mice spleens and transferred into nude mice via intravenous injection respectively. Experimental FK was induced by intrastromal injection of Candida albicans or Aspergillus fumigatus spores into the corneas one day after reconstitution. Development of corneal disease was measured by scoring the lesions in the corneas. We found that while immunocompetent Balb/c mice developed typical FK after pathogen challenge, T-lymphocytes deficient nude mice did not show any sign of microbial infection after challenge with same amount of fungal spores. Pre-infection reconstitution of nude mice with 2x10^7 T lymphocytes, and not with same number of B lymphocytes nor with 0.66x10^7 T lymphocytes, recovered the susceptibility of the nude mice to FK, and the disease in T cells-reconstituted nude mice were much more severe than in similarly infected Balb/c mice. We conclude that T lymphocytes above a threshold amount are essential in the initiation of keratitis when corneas are insulted by fungi, and T lymphocytes play a dual role in the pathogenesis and prognosis of fungal keratitis.
Elevated Bcl2 expression in mice decreases CD4 lymphocyte apoptosis and facilitates clearance of Pneumocystis in mice (40.6)
Authors: Opata Michael , Hollifield Melissa , Garvy Beth
Published: January 2023

Lymphocyte and TNF deficiency leads to susceptibility to opportunistic infections such as Pneumocystis carinii (PC). This strongly indicates that protection against the fungal pathogen PC is dependent upon B cells, T cells and TNF. Although it has been documented that macrophage derived TNF is essential for PC clearance, the role played by TNF produced by lymphocytes has not been addressed. Furthermore, the functional role of B cells in the T cell-mediated protection against PC Pneumonia (PCP) has not been determined. In this study we report that B cells produce TNF in response to PC predominantly in the TBLN. B cells enhanced expansion of CD4 T cells and secretion of TNF in the lungs of adoptive transfer recipients and hence clearance of the organisms. Importantly, the presence of B cells resulted in upregulation of Bcl2 in CD4 T cells and down regulation of Fas and Fas Ligand consistent with a decrease in apoptotic cells as determined by annexin V staining. Lastly, we observed that T cell-derived TNF is critical for PC clearance. Taken together, our data suggest that B cells are essential for CD4 T cell survival and increased expansion leading to protection against PCP in mouse models.
T cells activation and cytokines balance in active and cured tuberculosis (40.7)
Authors: Rodrigues Virmondes , Silva Marcos , Castellano Lucio , Machado Juliana , Figueiredo Amanda , Oliveira Rafael , Faria Gladstone , Pereira Sanivia , Rodrigues Denise
Published: January 2023

Tuberculosis is still a major health problem around the world and the understand of immune response against Mycobacterim tuberculosis is necessary for the development of new strategies for control disease progression. Activation of CD4+ and CD8+ T-cells by flow cytometry and the levels of cytokines in culture supernatants of PBMC from patients with active tuberculosis (n=25) comparing with individuals after successful treatment of tuberculosis (n=22). The activation of CD4+ but not CD8+ T cells was significantly higher in the tuberculosis-treated patients (p=0.04). The levels of IFN-γ and TNF-α were significantly higher among tuberculosis-treated subjects (p=0.005, p=0.034). The levels of cytokines involved in Th-2 and Th-17 axis did not differ among groups. We observed that the levels of IFN-γ and TNF-α were augmented only in subjects after more than 12 months of treatment (p=0.03 and p=0.01). Also, the huge production of Th1 cytokines by cured subjects was followed by an elevation in IL-10 production (p=0.03). Clinical cure due to treatment in tuberculosis was followed by the production of Th1 cytokines, although this recovery does not occur immediately after therapy ending. Th1 cytokine overproduction in healing process is followed by an elevation in IL-10 levels, indicating that concomitantly with elevation of pro-inflammatory cytokines there is a modulation of an immune response regulation-related cytokine.
The role of CD8+ T cells in cutaneous leishmaniasis caused by Leishmania braziliensis (40.9)
Authors: de Oliveira Camila , Novais Fernanda , Santiago Rômulo , Brodskyn Claudia , Barral-Netto Manoel , Barral Aldina
Published: January 2023

The leishmaniases are a group of diseases that affect millions of people worldwide. In the present study, we investigated the participation of CD8+ T cells during infection with L. braziliensis, the main etiolological agent of mucocutaneous leishmaniasis. Following inoculation of L. braziliensis into the ear dermis of BALB/c mice, we observed a high proportion of CD8+CD25+, at the time of lesion appearance, whereas CD8+CD69+ T cells were found at a greater proportion in the draining lymph nodes, at this same time point. Regarding cytokine production, a high percentage of CD8+IFN-g+ cells was detected as early as two weeks post infection, at the ear dermis. The same kinetics was observed for expression of CD107b. Interestingly, presence of IL-4+ and IL-10+ T cells was also detected during the course of infection in the ear dermis. Parasite load in β2-m deficient mice was smaller than in wild type (WT) mice. However, transfer of CD8+ cells obtained from L. braziliensis-primed mice induced the control of lesion development upon transfer to infected mice. These data indicate that CD8+ T cells may play a protective role in cutaneous leishmaniasis caused by L. braziliensis.
Locating the PTPN22 Gene in Families with Multiple Diagnosis of Autoimmune Disorders (41.5)
Authors: Lee Diane
Published: January 2023

While working at NIAID, I examined the PTPN22 gene, which encodes for the lymphoid protein tyrosine phosphatase (PTP), and has been shown to be associated with susceptibility to autoimmune disorders. PTP functions in a coordinated manner with protein tyrosine kinases in the regulation of signaling responses. Kinases are involved with controlling the amplitude of a signaling response and phosphatases are important for controlling the rate and duration of the response. From this laboratory experience, I developed an immunology unit for high school molecular biology and biotechnology classes. This is an intensive month long unit which introduces the first and second lines of defense in the immune system. This unit incorporates routine laboratory practices, such as polymerase chain reaction or PCR, restriction digest and analysis to identify individuals’ susceptibility towards autoimmune diseases. Upon completion of this unit, students will gain an understanding of the immune system through the PTPN22 allele and its effects on autoimmune diseases. The concluding unit activity has students study a family pedigree chart with multiple autoimmune diseases. The PTPN22 gene is isolated from the DNA of non-affected members from this pedigree chart, through the process of PCR. Using restriction digest and analysis, individuals’ DNA is tested to determine the presence of the PTPN22 allele and to establish autoimmune disease susceptibility.
Killer Proteins: Development of a bacteriocidal assay to study the role of complement in killing bacteria. (41.8)
Authors: Long John , Farrar John
Published: January 2023

Complement is an important component in the innate immune system’s defense against bacterial infections. In this unit, we teach basic principles of innate immunity by focusing on the biochemical nature of the complement cascade and its role in directly killing bacteria. We have developed a classroom curriculum that combines didactic course material with a novel laboratory exercise to help students understand the concept of complement in immunology. We have developed a bacteriocidal assay that students will perform to demonstrate the ability of complement to kill bacteria. This laboratory exercise, and subsequent background discussions, will introduce two key aspects of innate immunity. First, complement is a natural group of proteins secreted predominately by the liver which is an effective bacteriocidal agent. Second, complement and antibodies work harmoniously together to recognize and eliminate bacteria as a bridge to adaptive immunity. Students will be able to study the effects of both complement alone and complement plus antibody complexes in this bacteriocidal experiment. An interactive, hands-on laboratory activity enables students to quantify bacterial cell death with a colony forming unit assay of E.coli . Through this exercise, students will be able to focus on basic principles of innate immunity, experimental design and execution, and data analysis.
Increased resistance against MRSA-infection in immunocompromized hosts by injection of IL-12 producing cells. (42.10)
Authors: Saito-Taki Tatsuo , Suka Junko , Onogawa Tsuyoshi , Kitasato Hidero
Published: January 2023

Methicillin-resistant Staphylococcus aureus (MRSA) induces lethal infections at Health-care associated infection in immunocompromized hosts. Since MRSA shows multiple drug resistande, treatments by chemotherapeutic agents are hard to recover from illness. Murine fetal fibroblast cells C57/BL6 (C57/hIL-12) transfected with human IL-12 gene was established and the hIL-12 concentrations in peripheral bloods of hIL-12 were measured in normal beige femal mice and immunodefect beige male mice after ip injection of 1 x 106 of C57/hIL-12 cells and detectable dose of hIL-12 were detected at 12 hr. after injection. Concomitant injections of MRSA and hIL-12 could not induce any protective effects for beige male mice, but preinjection of hIL-12 before 48 hr from MRSA infection induced significant protective effects compared to concomitant injections. These results suggest that 48 hr is critical duration to restore the immune responses of immunodefect beige mice by activating cytokine network via IL-12.
The significance of TLRs in Brucella mediated dendritic cell activation in vitro and pulmonary clearance in vivo (42.16)
Authors: Surendran Naveen , Hiltbold Elizabeth , Sriranganathan Nammalwar , Heid Bettina , Boyle Stephen , Makris Melissa , Zimmerman Kurt , Witonsky Sharon
Published: January 2023

Brucellosis is a worldwide zoonoses affecting 500,000 people annually with no approved human vaccines available. Live attenuated B. abortus vaccine strain RB51 protects cattle through CD4 and CD8 T-cell mediated responses. However, there are concerns regarding its efficacy and use as a live vaccine in people. Therefore, identifying how Brucella vaccines stimulate innate and adaptive immunity is critical to enhancing vaccine efficacy. Brucella stimulate dendritic cells (DCs) through Toll-Like Receptors (TLRs) 2, 4 and 9. In this study, to identify how rough vaccine strains stimulate DC activation and function in vitro, we used vaccine strain RB51 versus pathogenic strain 2308 to stimulate bone marrow derived dendritic cells (BMDCs) from TLR2, 4 or 9 knockout (KO) and wild type BALB/c mice. Since inhalation of infected aerosols is one of the most common routes of exposure, in vivo clearance of strain RB51 compared to strain 2308 from intranasally (IN) infected TLR KO vs. control BALB/c mice was assessed. We determined that strain RB51 induced significant (p≤0.05) DC activation compared to strain 2308 which was TLR independent. However, strain RB51 induced TNF - α production was TLR2 and TLR9 dependent and IL-12 production was TLR2 and TLR4 dependent. TLR4 KO mice had significantly (p≤0.05) delayed pulmonary clearance of strain RB51 in vivo at day 14 post infection. Based on these data, future studies will focus on enhancing the protective ability of strain RB51.
Organization of bovine lambda light chain genes on chromosome 17 (43.14)
Authors: Pasman Yfke , Saini Surrinder , Smith Elspeth , Kaushik Azad
Published: January 2023

Twenty five Vλ genes, seventeen being functional Vλ, corresponding to the three Vλ gene families were identified upon complete characterization and physical mapping of bovine λ-light chain locus on chromosome 17. The bovine λ-light chain locus spans 412 kbp where the Vλgenes are located 23.7 kbp 5’ of four Jλ-Cλ units. The Vλgenes are organized in three subclusters separated by two large introns (126.8 and 138.3 kbp). The predominantly expressed Vλ1 genes are present in the two 5’ sub-clusters, while most Jλ-proximal Vλ sub-cluster comprises rarely expressed Vλ2 and Vλ3 genes. The Jλ3-Cλ3 unit was found predominantly expressed, though Jλ2 and Jλ3 have identical recombination signal sequences (RSS). The Cλ1 and Cλ4 are pseudogenes and their Jλ-Cλ units are not expressed. Endogenous counter selection for Jλ2 encoded CDR3 and framework 4 regions are likely the cause for the predominant expression of Jλ3-Cλ3 genes over Jλ2-Cλ2. The preferential expression of Vλ1 genes in the bovine Immunoglobulin repertoire seems to be influenced by the composition of RSSs. Polymorphism at the λ-light chain locus is responsible for differences in the genomic complexity between Hereford and Holstein cattle. Despite more potential germline encoded combinatorial diversity, the λ-light chain repertoire in cattle is predominantly encoded by Vλ1- Jλ3-Cλ3 recombinations. (Supported by NSERC Canada)
Activation of bovine NK cells through Toll-like receptors (43.2)
Authors: Mayer Sandra , Estes Don , Golde William
Published: January 2023

Toll like receptors (TLRs) play a critical role in the immediate detection and control of infection in mammals and are important for initiating a series of signaling events which result in proinflammatory gene expression. Many of these receptors are type I transmembrane proteins that belong to the IL-1 receptor family. There are 13 TLRs already identified in mammals, 10 in humans, 10 in bovine and 13 in mice. The TLRs main functions include secretion of type I interferon, proinflamatory cytokines, chemokines and expression of co-stimulatory molecules. The aim of this study was to verify intracellular perforin and IFN-γ production, granzyme and perforin transcription and extra cellular IFN-γ secretion in bovine NK cells treated with TLRs ligands such as TLR2, TLR3, TLR4, TLR5 and TLR9. Our preliminary results demonstrated that flagellin (6/6) and PPD (5/6) are the most inducers of intracellular IFN-γ production followed by LPS (4/6) and CpG (4/6). When we analyzed the intracellular perforin production we verified an increase in the cells treated with CpG (4/6) or LPS (3/6). The IFN-γ secretion in the supernatant was analized by a capture ELISA. The Poly I:C had the bigger impact in IFN-γ secretion followed by CpG and LPS which caused a small increase, while PPD and flagellin did not have any effect in IFN-γ secretion. These results suggest that bovine NK cells might be activated by these TLRs ligants, otherwise further studies are needed to elucidate this hypothesis.
Enhancement of Protein Immunogenicity via Synthesis of a Protein-LPS Chimeric Antigen (43.23)
Authors: Ye Jianmin , Kaattari Stephen
Published: January 2023

Typically, while LPS (TI-1) antigens elicit a rapid and robust primary response in teleost fish; they do not provide for good memory. On the other hand protein (TD) antigens, while excellent in priming for memory require much more time for producing a LPS-comparable primary response. This situation is problematic in aquaculture, wherein it is often only feasible to apply a single immunization. To investigate whether a chimeric antigen can produce an accelerated primary response to protein epitopes, while priming for memory, LPS was conjugated to a protein antigen (KLH). The effectiveness of this chimeric antigen (TNP-KLH--LPS) in enhancing an anti-protein response was demonstrated by comparison to protein alone (TNP-KLH) via cellular (elispot) and molecular (titer and affinity) assessments. This enhancement could be due to the increase of ASC number, their increased secretory capacity, or to an altered distribution of ASCs within the body. Furthermore, the chimeric antigen, as opposed to TNP-LPS, appears to elicit a number of higher affinity clones, similar to the protein antigen, which could provide highly specific memory response. Taken together, our results indicate that the chimeric immunogen significantly enhanced protein immunogenicity, which may significantly improve responses to viral subunit vaccines for farmed fish.
Transient Receptor Potential Melastatin 2 (TRPM2) is a Calcium Release Channel Essential for Chemotaxis of Dendritic Cells (44.17)
Authors: Sumoza-Toledo Adriana , Lange Ingo , Bhagat Harivadan , Cortado Hanna , Mori Yasuo , Penner Reinhold , Partida-Sanchez Santiago , Fleig Andrea
Published: January 2023

Chemokines induce Ca2+ signaling and chemotaxis in dendritic cells (DCs) but the molecular players involved in shaping the changes in intracellular Ca2+ remain to be characterized. Using siRNA and knock-out mice, we show that in addition to IP3-mediated Ca2+ release and store-operated Ca2+ entry the transient receptor potential melastatin 2 (TRPM2) channel contributes to Ca2+ release, but not Ca2+ influx in mouse DCs. Consistent with this observation, TRPM2 expression in DCs cells is restricted to endolysosomal vesicles, whereas in neutrophils the channel localizes to the plasma membrane and does not participate in Ca2+ release. While TRPM2 is not involved in DCs maturation, TRPM2-deficient DCs show severely compromised chemotaxis regardless of maturation stage. This is due to perturbed Ca2+ signaling manifested through suppression of specific TRPM2-mediated Ca2+-release events and an additional secondary modification of store-operated Ca2+ entry. DCs deficient in both TRPM2 and inositol-1,4,5-trisphosphate receptor signaling (InsP3R) lose their ability to perform chemotaxis entirely. These findings confirm ADPR as a novel second messenger and highlight TRPM2 as a key player regulating DCs chemotaxis through its function as Ca2+ release channel.
Staphylococcus aureus Biofilm-Mediated Infections: Characterization of the Host Adaptive Immune Response and its Role in Chronic Infection (45.2)
Authors: Prabhakara Ranjani , Harro Janette , Harris Megan , Leid Jeff , Costerton J. , Shirtliff Mark
Published: January 2023

Staphylococcus aureus is a common cause of prosthetic implant infections, which can become chronic due to the ability of S. aureus to grow as a biofilm. Little is known about adaptive immune responses to these infections in vivo. We hypothesized that S. aureus elicits inflammatory Th1/Th17 responses, associated with biofilm formation, instead of protective Th2/Treg responses. A previously developed mouse model of prosthetic implant infection was modified to produce a long term biofilm infection and used to determine chronic infection rates, Treg frequency, and local cytokine levels in B6 and BALB/c mice. Chronic infection levels in STAT6 KO mice (BALB/c background), BALB/c mice receiving anti-CD25 MAb, and B6 mice receiving anti-IL-6 or anti-IL-12 p40 MAb were also assessed. Local S. aureus implant infection was chronic in 100% of B6 mice, whereas BALB/c mice more effectively cleared the infection and demonstrated higher levels of local Th2 cytokines and Tregs. STAT6 KO mice lost the ability to effectively clear an S. aureus implant infection, as did BALB/c mice receiving anti-CD25. In contrast, B6 mice treated with anti-IL-12 p40 were able to more effectively clear the infection. These results indicate that Th2/Treg responses are mechanisms of protection against chronic S. aureus implant infection, as opposed to Th1/Th17 responses, which may play a role in development of chronic implant infection. Our results also suggest that anti-IL-12 p40 MAb may be used therapeutically.
HIV-1-specific exosome targeted CD8+ T cell Vaccine. (45.24)
Authors: Hebbandi Nanjundappa Roopa , Xiang Jim
Published: January 2023

Severe immuno-suppression due to immuno-tolerance and CD4 T cells depletion is the distinguishing feature of HIV-1 infection. Although highly active antiviral therapy (HAART) is available to halt progression of AIDS, it has its own inherent defects, including incomplete protection with associated side effects, and economical concerns. In addition, most of the currently available vaccines fail to induce effective CD8 CTL against HIV-1 in absence of CD4 T cells. Previously, our laboratory showed the efficacy of novel exosome (EXO)-targeted CD4 T cell vaccine (aTexo) with uptake of ova-specific dendritic (DC)-cell-released exosomes in the absence of CD4 T cells using highly metastasizing tumor model. In the present study, we find that aTexo vaccine prepared from active CD8 T cells is capable to induce CD8 CTLs, and its effect is mediated through IL-2 secretion, acquired pMHC I complexes, and partially on CD80 and CD40L costimulators. We have constructed an adenovirus-gp120 vector, which will be used to harvest gp-120-specific exosomes following DC infection, and established various tumor cell lines by transfecting gp-120 gene. We propose to generate gp-120-specific aTexo vaccine and test its efficacy in the presence or absence of CD4 T cells. These studies may significantly impact the development of novel therapeutic vaccines against HIV-1 infection.
Anti-Inflammatory Activity of IVIG Protects Against HSV Encephalitis. (45.26)
Authors: Chandran Ramakrishna , Shen YueWei , Cantin Edouard
Published: January 2023

Susceptible mouse strains like 129 mice develop HSV encephalitis (HSE) characterized by large focal inflammatory CNS lesions associated with massive accumulation of monocytes and neutrophils, resulting in rapid onset of death. In contrast, regulated CNS inflammatory responses protect resistant C57BL/6 mice against HSE. Disease progression was not affected by early inhibition of HSV replication consistent with immune mediated pathology and not virus cytopathology, causing death. Intravenous immunoglobulin (IVIG) possesses potent anti-inflammatory activities and is highly effective in treating various inflammatory diseases. We report here that IVIG protects against fatal HSE by inhibiting CNS inflammation through a mechanism(s) independent of HSV antibody. Both sialylated and non-sialylated but not deglycosylated IgGs mediated protection. IVIG treatment profoundly altered host immune responses, promoting lymphocytic rather than monocytic infiltration of the CNS accompanied by phenotypic alteration of macrophages and a dramatic expansion of CD4+ Tregs that accumulated in peripheral lymphoid organs. However the Tregs were not solely responsible for inhibiting inflammation. IL-10 secreted by APCs was primarily responsible for suppressing CNS inflammation. Despite controlling virus replication IVIG treated IL-10 deficient mice were unable to control CNS inflammation and succumbed eventually. In addition, FcγR2b was critical for IVIG’s immunomodulatory activity.
SIVmac251 infection of Rhesus Macaques destroys secondary lymphoid tissue architecture and depletes naïve T cell populations (45.9)
Authors: Zeng Ming , Wietgrefe Stephen , Schacker Timothy , Southern Peter , Reilly Cavan , Feldman Daniel , Carlis John , Silvestri Guido , Lifson Jeffery , Haase Ashley
Published: January 2023

The organized stromal cell network of secondary lymphoid tissue constitutes a microenvironment that plays a critical role in naïve T cells homeostasis, by providing survival factors such as interleukin-7 (IL-7). We previously showed that collagen deposition and damage to the lymphatic tissue niche in the course of HIV-1 infection contributes to CD4 T cell depletion and limits immune reconstitution with antiretroviral therapy. However, the mechanisms behind it have yet to be defined. Here we show in the SIV-rhesus macaque (Macaca mulatta; RMs) model, that collagen deposition and progressive loss of the stromal cell network limit both access and the source of interleukin-7 on which survival particularly of naïve T cells depends, and, consequently, greatly increased apoptosis results in progressive depletion of both naïve CD4 and CD8 T cell populations. We further show that the loss of the network and T cell depletion are interrelated by the stromal cell network’s dependence on lymphotoxin-beta produced by T cells. Thus, T cell depletion is a multiplicative and progressive pathological process involving access and interdependencies of T cells and supporting lymphatic tissue architecture.
Vaginal herpes simplex virus type 2 infection of mice elicits perforin-dependent immunopathologic tissue damage (46.10)
Authors: Cherpes Thomas , Vicetti Miguel Rodolfo , Kant Jeffrey , Phillips Jaclyn , Schreiner Robert , Harvey Stephen
Published: January 2023

Tissue injury and disease are often caused by host responses to invading pathogens rather than by the pathogen itself. To better understand the mechanisms responsible for immunopathogenic host responses, mice vaginally infected with herpes simplex virus type 2 (HSV-2) were used to model a viral pathogen capable of producing extensive collateral host tissue damage. We observed vaginal HSV-2 infection elicited intense local production of numerous proinflammatory cytokines and chemokines including IFN-gamma, IP-10, and MIP-1 alpha. Beginning 2 dpi, this inflammatory milieu was responsible for recruitment of large numbers of inflammatory monocytes, NK cells, and NKT cells into infected vaginal mucosa. As characterized by their expression of high levels of both perforin and granzyme B, infiltration of these highly activated NK and NKT cells was highly correlated with the onset of genital pathology. Perforin deficient mice cleared primary infection with similar kinetics as wildtype controls while exhibiting significantly decreased levels of genital pathology. The perforin deficient mice were, however, incapable of generating immune responses that prevented lethal dissemination of the virus to the central nervous system. Our data demonstrate a previously unrealized contribution of perforin-mediated mechanisms for HSV-induced tissue damage and may prove useful for exploration of therapeutic interventions that decrease immunopathology without compromising viral clearance.
Secretory IgA responses induced by rice-based oral cholera toxin B subunit vaccine are solely responsible for antibody-mediated long-standing cross-protection against Vibrio cholerae - and enterotoxigenic Escherichia coli -induced diarrhea (46.3)
Authors: Tokuhara Daisuke , Yuki Yoshikazu , Nochi Tomonori , Kodama Toshio , Mejima Mio , Kurokawa Shiho , Takahashi Yuko , Nanno Masanobu , Takaiwa Fumio , Honda Takeshi , Kiyono Hiroshi
Published: January 2023

Introduction: We investigated whether the cholera toxin B subunit (CTB)-specific secretory IgA (SIgA) induced by rice-based oral vaccine expressing CTB (MucoRice-CTB) gives long-term protection against V. cholerae - and enterotoxigenic Escherichia coli (ETEC)-induced diarrhea. Methods: Mice were orally immunized with MucoRice-CTB, which stored at room temperature for 3 years, and then evaluated for induction of SIgA-mediated protective immunity against CT or heat-labile enterotoxin (LT). The role of CTB-specific SIgA was also examined in polyimmunoglobulin receptor (pIgR)-deficient mice. Long-term immune memory was evaluated 6 months after final primary immunization. In vivo V. cholerae (El Tor O1 Inaba) and ETEC challenge was performed in the intestinal loops of immunized mice. Results: Oral MucoRice-CTB immunization provided SIgA-mediated protection against LT- or CT-induced diarrhea. This protection was impaired in pIgR-deficient mice. The vaccine offered long-lasting protection against toxin-induced diarrhea (for at least 6 months after primary immunization), and a single booster immunization extended the length of protective immunity for at least 4 more months. In the intestinal loop assay, MucoRice-CTB vaccination inhibited diarrhea in the face of V. cholerae and ETEC challenge. Conclusion: MucoRice-CTB is an effective cold-chain-free oral vaccine inducing CTB-specific SIgA-mediated long-term protection against V. cholerae - or ETEC-induced diarrhea.
Mucosal S-IgA Abs is required for complete immunity to Streptococcus pneumoniae (46.6)
Authors: Fukuyama Yoshiko , King Janice , Kobayashi Ryoki , Gilbert Rebekah , Hollingshead Susan , Briles David , Fujihashi Kohtaro
Published: January 2023

We investigate whether pneumococcal surface protein A (PspA)-specific secretory (S)-IgA antibodies (Abs) induced by nasal vaccine is necessary for the prevention of streptococcal colonization and their clearance. IgA deficient (IgA-/-) and control mice were nasally immunized four times at weekly intervals with PspA plus flt3 ligand plasmid (pFL). One week after the last immunization, PspA-specific S-IgA and IgG Ab as well as Th1- and Th2-type cytokine responses by CD4+ T cells were determined. Further, mice were challenged with Streptococcus pneumoniae (EF3030) via the nasal route. Five days after challenge, nasal washes (NWs) and nasal passages (NPs) were collected and the numbers of bacterial colonies were determined. Mice nasally immunized with PspA plus pFL showed significantly increased levels of Th2 cell-mediated PspA-specific S-IgA and IgG Ab responses in NWs and plasma when compared with naïve mice. Thus, these vaccinated wild type mice showed essentially no bacteria in the nasal cavity. Although IgA-/- mice given nasal PspA plus pFL had significantly high levels of PspA-specific IgG Abs, high numbers of bacteria were detected in NWs and NPs. Further, nasal PspA plus pFL effectively reduced pre-existing S. pneumoniae in the nasal cavity of wild-type, but not IgA-/- mice. These results show that PspA-specific S-IgA Abs play important roles in the prevention of S. pneumoniae colonization and clearance. Supported by NIH grants AG025873 and DE12242.
TLR2 Regulation of Oral Keratinocyte Driven Mucosal Homeostasis in the Context of Diabetes Mellitus (47.11)
Authors: Wallet Shannon
Published: January 2023

Oral epithelium contributes to innate immunity and plays an important role in immune tolerance/homeostasis. Disruption of this homeostasis can result in initiation and/or exacerbation of localized inflammation that secondarily causes/worsens systemic diseases. Toll-like receptor 2 (TLR2) is one molecular pattern receptor that under cell- and/or environment-specific conditions can induce either inflammatory or regulatory immune responses. Hyperglycemia, a sequela of diabetes mellitus, has multiple profound effects on the local innate responses, which drives cyclical pathogenesis where elevated glucose disrupts mucosal homeostasis, exacerbating oral inflammation that in turn compounds systemic manifestations of diabetes. We evaluated the innate immune responses of oral keratinocytes (HOK) under homeostatic and hyperglycemic conditions. Our results demonstrated that TLR2 homo-dimerization resulted in an immunoregulatory response, and that this maintenance of tolerance is interrupted under hyperglycemic conditions. We have also demonstrated that regulation of this immunoregulatory response involves expression and phosphorylation of TLR signaling intermediates regulated by multiple miRNA. We propose that HOK regulation is multi-factorial and our model will facilitate development of therapies designed at restoring oral homeostasis to stop the vicious cycle linking systemic diseases such as diabetes with disruption of mucosal homeostasis and exacerbation of the systemic disease.
Exacerbation of DSS induced colitis by localized delivery of IFN-β secreted by Lactobacillus acidpohilus (47.6)
Authors: McFarland Adelle , Savan Ram , Wagage Sagie , Addison Augustina , Young Howard
Published: January 2023

There have been conflicting reports of the role of Type I interferons in gut inflammation and inflammatory bowel diseases. While in some patients with active ulcerative colitis (UC), treatment with Type I interferons induced remission of symptoms, other reports have documented the spontaneous occurrence of UC in multiple sclerosis and chronic hepatitis C patients receiving IFN-β therapy. In order to evaluate the potential for IFN-β as a therapeutic for IBD, we developed a delivery system involving probiotic bacteria. Treatment of healthy mice with Lactobacilli secreting IFN-β results in increased IFN-γ positive cells within the gut as well as a reduction in the percentage of T-regulatory cells. When mice were pretreated with Lactobacilli secreting IFN-β prior to induction of acute DSS colitis, we observed more weight loss and a worsening of intestinal thickening/shortening. In addition, there was an increase in the percentage of Th17 cells, as well as an increase in the production of TNF-α by colonic tissue. This study demonstrates that localized delivery of IFN-β by probiotic bacteria results in an exacerbation of DSS induced colitis in a preventative model.
Critical role for the NLRP3 inflammasome in colitis and colitis associated colorectal cancer (47.9)
Authors: Zaki Mohammad , Kanneganti Thirumala-Devi
Published: January 2023

Mutations in NOD-like receptor (NLR) Nlrp3 cause a variety of rare autoinflammatory disorders. Decreased expression of the Nlrp3 was recently associated with susceptibility to Crohn's disease. Nlrp3 interacts with the adaptor protein ASC to activate caspase-1 in the inflammasome, a protein complex responsible for the maturation and secretion of IL-1β and IL-18. However, the role of Nlrp3 in colitis has not been characterized. Here, we show that mice deficient for Nlrp3 or the inflammasome effectors ASC and caspase-1 are highly susceptible to dextran sodium sulfate (DSS)-induced colitis. Defective inflammasome activation leads to loss of epithelial integrity, resulting in systemic dispersion of commensal bacteria, massive leukocyte infiltration and increased chemokine production in the colon. To explore the possibility that increased inflammation in inflammasome-deficient mice may lead to tumorigenesis, we developed colorectal tumor model using azoxymethane followed by DSS-induced chronic colitis. Nlrp3- and caspase-1-deficinet mice showed an increased incidence and multiplicity of tumors compared to those of wild-type. Antibiotics treatment ameliorated clinical readouts of colitis and tumorigenesis in Nlrp3-deficient mice, demonstrating the role of commensal microflora in aggravating colitis severity. Therefore, the Nlrp3 inflammasome is critically involved in the maintenance of intestinal homeostasis and protection against colitis and colorectal tumorigenesis.
B cells in chronically inflamed skin. (48.10)
Authors: Geherin Skye , Fintushel Sarah , Hay John , Gudrun Debes
Published: January 2023

B cells infiltrate extralymphoid tissues in many chronic inflammatory diseases, including cutaneous manifestations of autoimmunity as well as infection. However, the phenotype and function of B cells in the skin remains poorly understood. In a granuloma model of cutaneous inflammation in sheep and mice, B cells increase during the chronic phase of inflammation in both percentage and absolute number in the skin and skin-draining afferent lymph. B cells in the skin and skin-draining lymph are a heterogeneous population that is phenotypically distinct from B cell populations in the spleen and lymph nodes. Many skin B cells exhibit an ‘innate-like’ phenotype co-expressing IgM and CD11b. Furthermore, skin B cells have increased MHCII and CD80/86 expression compared to lymph node or splenic B cells, suggesting that they are well-suited for T cell activation at the site of inflammation. Additionally, skin B cells are not responsive to ligands of chemokine receptors typically associated with skin homing. The data support a model in which innate-like B cells use alternative chemokine receptors to migrate to the skin where they support inflammatory or protective immune responses.
Lung CD4+ T cell receptor AV2S3+ T cells in sarcoidosis respond to the mycobacterial protein mKatG (48.6)
Authors: Wikén Maria , Ostadkarampour Mahyar , Willett Matthew , Chen Edward , Eklund Anders , Moller David , Grunewald Johan , Wahlström Jan
Published: January 2023

Background Sarcoidosis is an inflammatory disease in which T cell responses have been found against the mycobacterial protein mKatG. Our aim was to study the ability of lung and blood CD4+ or CD8+ T cells, and in particular T cell receptor (TCR) AV2S3+ CD4+ T cells, to respond with cytokine production to mKatG. Methods Bronchoalveolar lavage (BAL) fluid and peripheral blood were obtained from 13 HLA-DRB1*0301+ patients, who recover spontaneously, all with lung-accumulation (>10.5% of CD4+) of AV2S3+ T cells. Using flow cytometry and intracellular cytokine staining lung and blood T cells were studied with regard to IFNγ, TNF and IL-2 production, after mKatG or purified protein derivate (PPD) stimulation. Results Lung CD4+ T cells responded with IFNγ, TNF and IL-2 production after mKatG or PPD stimulation, whereas CD8+ T cells responded with IFNγ production, and only to mKatG. AV2S3+ T cells responded with IFNγ secretion to mKatG to a significantly higher extent than AV2S3- T cells, whereas the opposite was seen for PPD. Blood CD4+ T cells responded with IFNγ and TNF production after mKatG or PPD stimulation. Stimulation with mKatG generated a large fraction of single IFNγ-producing cells, whereas PPD stimulated to a more pronounced multifunctional profile with IFNγ/TNF or IFNγ/IL-2 secretion. Conclusion A specific mycobacterial antigen, mKatG, triggers a subset of the AV2S3+ T cells, indicating a possible role for these cells in eliminating an offending antigen in sarcoidosis.
IL-6 and IL-1α together, but not alone, are key modulators of regulatory T cell suppressive function: insights from computational and in vitro studies. (49.11)
Authors: Raimondi Giorgio , Ziraldo Cordelia , Pillai Mahesh , Manzoni Giulia , Kowalkowski Frank , Vodovotz Yoram , Thomson Angus
Published: January 2023

Modulation of the susceptibility of effector T cells to suppression by naturally-arising regulatory T cells (Treg) influences the development of autoimmune diseases, cancer, and transplant outcome. However, our understanding of the factors that underlie this modulation is limited. We aimed to identify the inflammatory mediators that are released by innate immune cells following their exposure to danger signals, and that reduce T cell susceptibility to suppression. A CFSE-based, in vitro mouse T cell activation system was employed to quantify Treg function and to measure the influence of the supernatant of dendritic cells sampled at multiple times after stimulation with the TLR4 ligand LPS. In parallel, 24 cytokines were quantified using Luminex™ in each sample tested. ‘Principal Component Analysis’ was utilized to assess the likely role of these cytokines with the extent of Treg suppression. This analysis suggested that IL-6, IL-1α, and TNFα were possible inhibitory mediators working in synergy. Using the CFSE dilution assay, we found that no single cytokine was capable of reducing Treg suppression. However, combinations of cytokines, in particular IL-6 and IL-1α, resulted in pronounced modulation of suppression. Our results suggest that combining computation with experimentation may help to dissect the complex milieu that determines T cell susceptibility to suppression, and specifically to identify the synergy between IL-6 and IL-1α as target for immunomodulatory therapies.
CD200Fc(Gly)6TGFβ suppresses transplant rejection and MLCs in vitro (49.15)
Authors: Gorczynski Reginald , Chen Zhiqi , Shivagnahnam Saaiis , Taseva Adriana , Wong Karrie , Yu Kai , Khatri Ismat
Published: January 2023

CD200 suppresses graft rejection after engagement of receptors (CD200R), via induction of Treg and production of TGFβ. We asked if a hybrid molecule linking a soluble form of CD200, CD200Fc, to murine TGFβ through a (Gly)6 linker would produce superior suppression to either CD200FC or TGFβ alone, or in combination. MLC cultures used BL/6 responder cells and irradiated BALB/c DCs, in the presence/absence of varying doses of CD200Fc or TGFβ, alone or in combination, or CD200Fc(Gly)6TGFβ. CD200Fc(Gly)6TGFβ produced suppression at 20-100-fold lower concentrations than all other reagents tested alone or together. When used in vivo to suppress rejection of BALB/c skin grafts in C57BL/6 mice, CD200Fc(Gly)6TGFβ also produced immunosuppression at 100-fold lower concentrations. Further studies suggest the mechanism(s) by which this novel molecule produces suppression involves increased numbers of (inducible) Foxp3+ Treg which cause antigen-specific suppression in MLCs in vitro. Using cells with targeted deletion of the main receptor for CD200 (CD200R1), and/or treated with lentiviral particles encoding shRNAs specific for TGFβRII, we showed that suppression depended on responder T cells with a functional TGFβRII, and DCs expressing CD200R1. Augmented inhibition of suppression by CD200Fc(Gly)6TGFβ using anti-CD200R2 in cultures from CD200R1 knockout mice confirmed a role for CD200R2 in suppression. We conclude that CD200Fc(Gly)6TGFβ may have clinical utility in vivo, or ex vivo.
1,25-dihyroxyvitamin D3 enhances the activity of the FOXP3 gene in human CD4+ T cells through directly binding to putative VDRE sites in the FOXP3 gene (49.2)
Authors: Kang Insoo , Kim Sang Hyun , Lee Won-woo , Kang Seong Wook , Lee Jin Soo
Published: January 2023

1,25-dihyroxyvitamin D3 (1,25(OH)2VD3) is known to have immune regulatory effects on T cells. However, it is unknown whether 1,25(OH)2VD3 can directly regulate the expression of the FOXP3 gene in human CD4+ T cells. We thus searched and found putative response elements for vitamin D receptor (VDR) in a highly conserved non-coding sequence (CNS) region of the human FOXP3 gene that encompassed +1,722 to +2,562 using NUBIscan algorithm. The presence of VDR and retinoid X receptor (RXR), which heterodimerizes with VDR, in the nucleus of human CD4+ T cells was detected by co-immunoprecipitation using biotinylated fragments of the CNS region that contained the putative vitamin D response elements (VDRE) sites. We next performed a chromatin immunoprecipitation (ChiP) assay to determine whether VDR directly bound to the sites. The results of this assay showed the binding of VDR to the putative VDREs of the CNS but not to the promoter of the FOXP3 gene. To test whether 1,25(OH)2VD3 could directly enhance the FOXP3 promoter activity, we conducted a reporter assay using a PGL3 vector containing the FOXP3 gene promoter with or without VDREs in the CNS of the FOXP3 gene. Indeed, 1,25(OH)2VD3 enhanced the promoter activity of the FOXP3 gene in the presence of VDREs. Our findings indicate that 1,25(OH)2VD3 up-regulate the FOXP3 gene expression in human CD4+ T cells through directly binding to VDREs in the CNS region of the FOXP3 gene that encompasses +1,722 to +2,562.
SPPL3 regulation of Ca2+ homeostasis modulates NFAT activation during antigen-receptor stimulation in lymphocytes (50.11)
Authors: Lew Stefanie , Pomerantz Joel
Published: January 2023

During T cell receptor signaling, activation of several transcription factors initiates new gene expression, leading to the generation of specific T cell responses. One important event in this pathway involves store-operated calcium entry (SOCE) through Ca2+-release-activated-Ca2+ (CRAC) channels, which activates the key transcription factor NFAT. Induction of this Ca2+-dependent signaling necessitates tight regulation of intracellular Ca2+ levels, and while the identification of STIM1 and CRACM1 has significantly helped elucidate how SOCE is achieved, other players in this pathway and alternative unknown pathways also controlling Ca2+ levels are not well understood. Here, we have used an expression cloning screen to identify signal peptide peptidase like 3 (SPPL3) as a novel activator of NFAT. Interestingly, SPPL3 is a polytopic membrane protein which localizes to the ER or Golgi compartment. Using Ca2+-sensitive dyes, we determined that SPPL3 overexpression elevated basal intracellular Ca2+ levels, dampened the thapsigargin-stimulated release of ER Ca2+ stores, and enhanced Ca2+ influx. SPPL3-mediated NFAT activation was blocked by the SOCE inhibitor SKF 96365 and a dominant negative CRACM1. By influencing intracellular Ca2+ levels, SPPL3 appears to be a novel regulator of calcium homeostasis that can positively modulate antigen receptor-mediated activation of NFAT.
The role of β2 integrins in host responses to intracellular bacterial infection in mice (50.27)
Authors: Bose Tina , Khanna Dr. Kamal , Lefrancois Dr. Leo
Published: January 2023

β2 (CD18) integrins (CD11a,b,c,d) are important adhesion molecules necessary for leukocyte migration, homing, and cell to cell interactions. CD18-deficiency in humans is characterized by recurrent bacterial infections and poor wound healing, due to defects in leukocyte activation and migration. However, the in vivo role of each of the individual β2 family members is poorly understood. CD11a, CD11b and CD11c are all upregulated on activated CD8 T cells. In order to study the potential role for β2 integrins in host defense, CD11a, CD11b and CD11c deficient and wildtype mice were infected with Listeria monocytogenes (LM). Surprisingly, the absence of the integrins had no effect on the expansion of antigen specific CD8 T cells in response to infection with LM. Although CD11b-/- and CD11c-/- exhibited normal effector functions, granzyme levels and effector cytokines were reduced in CD11a-/- CD8 T cells as compared to WT CD8 T cells post LM infection. CD11b and CD11c are also highly expressed by important antigen presenting cells such as macrophages and dendritic cells (DC). Therefore, we tested whether the absence of CD11c or CD11b compromised DC function. However, preliminary experiments indicated that upon infection, CD11b-/- and CD11c-/- DCs are capable of normal activation and migration. Overall, these results suggested that unlike CD11b and CD11c, CD11a may play an integral role in induction of optimal effector functions in CD8 T cells.