bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of needle-free vaccine: rice-based oral vaccine induced protective immunity against cholera toxin (41.4)
Authors: Nochi Tomonori , Yuki Yoshikazu , Takagi Hidenori , Yang Lijun , Masumura Takehiro , Mejima Mio , Nakanishi Ushio , Matsumura Akiko , Hiroi Takachika , Morita Shigeto , Tanaka Kunisuke , Takaiwa Fumio , Kiyono Hiroshi
Published: January 2023

Capable of inducing antigen-specific immune responses in both systemic and mucosal compartments without the use of syringe and needle, mucosal vaccination is considered ideal for the global control of infectious diseases. In this study, we developed a rice-based oral vaccine expressing cholera toxin B subunit (CTB). An average of 30 μg of CTB per seed was stored in the protein body, a rice storage organelle. When orally fed, rice seeds expressing CTB were taken up by the M cells covering the Peyer′s patches (PPs), inducing toxin-specific serum IgG and mucosal IgA antibodies with neutralizing activity. When expressed in rice, CTB was protected from pepsin digestion in vitro. Rice-expressed CTB also remained stable and thus maintained immunogenicity at room temperature for more than 1.5 years, meaning that antigen-specific mucosal immune responses were induced at much lower doses than were necessary with purified recombinant CTB. Because they would require neither needles nor refrigeration (cold-chain management), these rice-based oral vaccines offer a highly practical as well as cost-effective strategy for orally vaccinating large populations against mucosal infections, including those which may result from an act of bioterrorism.
Th2 polarizing helminth infection alters the immune response to an oral Salmonella-OVA vaccine (41.5)
Authors: Iweala Onyinye I. , Smith Donald W. , Urban Joseph F. , Nagler Cathryn R.
Published: January 2023

We have previously shown that enteric helminth infection acts as a mucosal adjuvant, priming for a Th2 biased response to soluble ovalbumin (OVA), a tolerogenic form of dietary antigen. An immunogenic response to orally administered OVA can be elicited by administration of OVA in the context of an oral vaccine. To examine whether chronic enteric infection can also influence the efficacy of oral vaccination we examined the response to a novel OVA-expressing oral Salmonella vaccine (Salmonella-OVA; Smith et al submitted) in both infected and non-infected mice. This live attenuated vaccine induces a Th1 biased mucosal and systemic OVA specific response. Infection of C57Bl/6 mice with the helminthic parasite Heligmosomoides polygyrus delayed and reduced the Th1 dependent serum OVA specific IgG2c response induced by two doses of oral vaccine. By contrast, helminth infection enhanced the Th2 dependent serum OVA specific IgG1 response. Helminth infection also altered mucosal and systemic OVA specific cytokine and chemokine responses. These data suggest that concomitant enteric helminth infection can significantly alter the immune response to oral vaccination. Since chronic enteric parasitic infections are endemic in much of the developing world, our observations have important implications for the testing and development of mucosal vaccines.

This work was supported by NIH grants DK 55678 and DK43551 and predoctoral training fellowships 5F31AI054229-05 and T32 A107529.
Small and large intestinal intraepithelial T lymphocytes show distinct dependency on sphingosine 1-phosphate (42.11)
Authors: Kunisawa Jun , Kurashima Yosuke , Higuchi Morio , Gohda Masashi , Ishikawa Izumi , Ogahara Ikuko , Kim Namju , Shimizu Miki , Kiyono Hiroshi
Published: January 2023

It is known that the composition of intraepithelial T lymphocyte (IEL) differs between small and large intestines, but the mechanism underlying that difference remains obscure. Here, we show that sphingosine 1-phosphate (S1P) plays a key role in regulating intestinal IEL trafficking into the small and large intestines. High levels of type 1 S1P receptor (S1P1) expression was noted on naïve IELs expressing CD4 or CD8αβ, which leads to their preferential migration into the large intestine. In contrast, recent thymic emigrants (RTEs), double-positive thymocytes, and double-negative thymic T cell-committed precursors use S1P-independent trafficking pathway into the intestine. The former two populations exclusively migrate into the small intestine, while the latter double-negative thymic T cell-committed precursors migrate into both the small and large intestines. Hence, down-regulation of S1P1 expression inhibited naïve IEL migration into the intestines but did not affect the migration of thymic IEL precursors. These data are the first to demonstrate that a lipid-mediated system determines whether IELs migrate to the small or large intestine.
Placental cytotrophoblast HLA-G5 is synthesized as a β2m-free heavy chain homodimer (42.13)
Authors: Morales Pedro J , Pace Judith L , Platt Jeralyn S , Hunt Joan S
Published: January 2023

Placental villous cytotrophoblast cells (vCTB) contain an unusual member of the HLA class I gene family, HLA-G5. Recently we showed that HEK293-derived recombinant HLA-G5 protein is predominantly composed of H:H homodimers. Here, we examined the HLA-G5 produced in primary vCTB. Determining the structure is critical because recombinant ectodomains of the inhibitory leukocyte Ig-like LILRB1 (ILT2) and LILRB2 (ILT4) receptors bind HLA-G dimers with higher affinity than monomers. Analysis of vCTB cell extracts by immunoblotting demonstrated that vCTB cell HLA-G5 H chain proteins are primarily disulfide bonded dimers. The H chains were not associated with β2m L chains; although β2m mRNA was identified by RT-PCR, immunoblots failed to detect β2m protein even when additional β2m mRNA was introduced. Furthermore, sequencing failed to reveal any abnormalities in the translational start codon of either endogenous β2m mRNA or introduced β2m mRNA, and EGF-induced syncytialization did not promote β2m in vCTB cells. The inability of vCTB cells to translate β2m messages in vitro was reflected in failure to detect β2m in vCTB cells in situ. Thus, vCTB cells may synthesize a disulfide-bonded, β2m-free homodimeric form of HLA-G5 due to an inability to synthesize β2m protein. This molecule could comprise a particularly effective tolerogenic molecule at the maternal-fetal interface.
Late Pregnancy Serum Factors Suppress T Cell Activation in Experimental Autoimmune Encephalomyelitis (EAE) (42.14)
Authors: Gatson NaTosha N , McClain Melanie , Gienapp Ingrid E , Powell Nicole , Shawler Todd M , Kithcart Aaron P , Whitacre Caroline C
Published: January 2023

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS thought to be due to an autoimmune response directed against myelin antigens. EAE is a commonly used animal model for MS and shares clinical, histopathologic and immunologic similarities with MS. Profound suppression of the MS and EAE relapse rate is observed during late pregnancy and is followed by a marked increase in disease severity postpartum. We have recently observed that histopathologic progression of EAE is diminished during late pregnancy and is exacerbated post partum. During EAE and late pregnancy, mice were found to have less mononuclear CNS infiltrates, demyelination and axon severing than virgin controls. The purpose of this study was to explore the hypothesis that decreased autoimmune disease during pregnancy is due to immunosuppressive factors present in the serum during late pregnancy. Female SJL/J mice were immunized for EAE using myelin proteolipid protein (PLP) 139–151. T cell proliferation assays were carried out using lymph node (LN) cells stimulated with PLP or anti-CD3 in the presence of 0–3% late pregnancy or virgin mouse sera. LN cells from age and gender matched naïve mice served as controls. Three percent late pregnancy serum suppressed both anti-CD3 and PLP stimulated T cell proliferation as compared to cells cultured with virgin mouse sera. These results demonstrate the broad suppressive potential of late pregnancy serum on T cell activation and may be responsible for amelioration of clinical signs of EAE during late pregnancy.

(Supported by NIH grants NS48316 and T32AI055411).
Immunomodulatory actions of Treg PPAR γ in the gut mucosa (42.3)
Authors: Hontecillas Raquel , Bassaganya-Riera Josep
Published: January 2023

Peroxisome proliferator-activated receptor (PPAR) γ activation has been implicated in the prevention of immunoinflammatory disorders, however the mechanisms of immune modulation by endogenous PPAR γ agonists remain unclear. We have used PPAR γ-deficient CD4+ T cells obtained from tissue-specific PPAR γ null mice (i.e., PPAR γ fl/fl; MMTV-Cre+) to investigate the role of endogenous PPAR γ on regulatory CD4+ T cell (Treg) function. Overall, we show that the transfer of purified PPAR γ null CD4+ T cells into SCID recipients results in enteric disease. To test the assertion that the deficiency of PPAR γ in Treg impairs their ability to prevent effector T cell-induced colitis, we performed co-transfer studies using PPAR γ null and PPAR γ-expressing CD4+ T cells. These studies demonstrate that PPAR γ-expressing, but not PPAR ? null Treg, prevent colitis induced by transfer of naïve CD4+ T cells into SCID recipients. In line with these findings, the production of IFN-γ by spleen and mesenteric lymph node-derived CD4+ T cells was down-regulated following transfer of PPAR γ-expressing, but not PPAR γ null, Treg. Furthermore, the loss of PPAR γ limited the expansion of Foxp3+ cells systemically and their accumulation in the colonic mucosa. In conclusion, our data suggest that endogenous PPAR γ activation represents a Treg intrinsic mechanism of downregulation of effector CD4+ T cell function and prevention of intestinal inflammation.
Role of NKT cells in the placenta and uterus of pregnant mice infected by Chlamydia trachomatis (42.5)
Authors: Kanellopoulos-Langevin Colette , Delarbre Christiane , Habbeddine Mohamed , Verbeke Philippe , Moutier René , Ojcius David
Published: January 2023

In order to study the role of maternal NKT cells against a Chlamydia infection during pregnancy, we have compared CD1d-deficient, C57Bl/6 (B6) mice to wild type controls, in the presence or absence of a Chlamydia infection. We have examined the expression of several molecules known to be important for NKT cell function, in the uterus and placenta. We focused our study on NKG2A, NKG2C and NKG2D receptors, their ligands Qa-1 and Rae-1 and their co-receptors CD94, DAP10 and DAP12. We also studied perforin, granzyme B, osteopontin and cytokines such as TNF alpha, interferon gamma and IL1 beta, Our results suggest that CD1d-restricted NKT cells do not play a major role in the control of infection, as the intensity and duration of infection were comparable in CD1d-deficient and wild type mice. However, we found that Chlamydia infection caused an increase of invariant NKT (iNKT) cells in the placenta and uterus of B6 mice, measured by the expression of their characteristic TCR Valpha14-Jalpha18 invariant chain. Correlatively, Chlamydia infection in CD1d-deficient mice lead to organ-specific modulations in the expression of several NKT cell markers, which differed from those observed in B6 mice.

Supported by NIH grant number 5 R21 A1060725-02, CNRS and Universities Paris 6 and 7.
CXCL9 and CXCL10 Expression are Critical in Resistance to Genital Herpes Simplex Virus Type 2 (HSV-2) Infection (43.15)
Authors: Thapa Manoj , Carr Daniel
Published: January 2023

The relevance of CXCL9 and CXCL10 expression in protection against genital herpes simplex virus type 2 (HSV-2) infection was investigated using mice deficient in CXCL9 (CXCL9−/−) or CXCL10 (CXCL10−/−) along with C57BL/6 wild type (WT) mice. Progesterone-sensitized mice infected with 2,000 pfu HSV-2 intravaginally were exsanguinated at times post infection (p.i.), and the vaginal tissue (VG), spinal cord (SC), and brain stem (BS) were removed and assayed for virus titer, leukocytes infiltration, and cytokine/chemokine content. CXCL10−/−, but not CXCL9−/− or WT mice were found to possess HSV-2 in the SC at day 3 following infection. By day 7 p.i., CXCL9−/− and CXCL10−/− mice harbored significantly more virus in the SC and the BS compared to WT mice. The increase in virus titer in the BS and SC of the chemokine deficient mice correlated with a decrease in NK cell mobilization to the nervous system at day 5 p.i. and CD8+ T cells mobilization at day 7 p.i. In addition, there was a noticeable increase in vaginal inflammation (measured by cytokine/chemokine levels and leukocyte infiltration) in the CXCL10−/− mice compared to the WT or CXCL9−/− mice. Antibody-mediated depletion of NK cells resulted in an increase in HSV-2 levels in the vagina, SC, and BS of wild type mice suggesting these cells contribute to resistance to infection.

This work supported by AI053108.
The Effects of Listeria monocytogenes Infection on Dendritic Cell Subsets (43.16)
Authors: Mitchell Latoya M. , Grayson Jason , Hiltbold Elizabeth M.
Published: January 2023

Dendritic cells (DC) are antigen presenting cells that link innate and adaptive immunity and have been shown to be a vital component for clearance and protection against Listeria monocytogenes (Lm) infection. Because of the role of DC in immunity against Lm and the varied functional capacities of DC subsets, our goal is to determine the impact of Lm infection on CD8α+/CD24+, myeloid, and plasmacytoid DC subsets found in Flt3L bone-marrow derived DC (BMDC) and mouse spleen.

Previously, we found that Flt3L BMDC undergo maturation upon infection with wild-type Lm as well as a Hly− mutant Lm that cannot enter the cytoplasm. The response in Flt3L BMDC to Hly− Lm is also dependent on signaling through the Toll-like receptor adaptor, MyD88. These data are in contrast to GM-CSF BMDC which only mature in response to cytoplasmic Lm. In vivo studies showed that the peak of costimulatory molecule upregulation following i.v. infection with wild-type Lm is 24 hours. Splenic DC, in contrast to Flt3L BMDC, did not increase surface costimulatory molecules in response to Hly− Lm. The frequency of splenic DC subsets following Lm infection and their ability to present antigen to T cells was also studied. In summary, our goal is to understand the capacity of different DC subpopulations to handle Lm infection and in future studies determine the role of each DC subset in establishing protective immunity. (NIH AI057770)
Lytic granule components are required for the maintenance of HSV-1 neuronal latency (43.35)
Authors: Knickelbein Jared E , Khanna Kamal M , Hendricks Robert L
Published: January 2023

Using a murine model of HSV-1 corneal infection, our data suggest that secretion of CD8 T cell lytic granules (LGs), in addition to IFN-γ, is required for the maintenance of viral latency within trigeminal ganglia (TG). First, HSV-specific CD8 T cells (HSV-CD8), which recognize an immunodominant HSV-1 glycoprotein B (gB498–505) epitope, surround neurons within latently infected TG and contain functional LGs, with GrB polarizing to the contact site with these neurons. HSV-CD8 use GrB and IFN-γ to block HSV-1 reactivation from latency in a non-redundant manner in ex vivo TG cultures, possibly due to a lack of IFN-γ receptors on some latently infected neurons. Like WT mice, Pfn and GrB KO mice establish latency in the TG by 8 days post-infection (dpi) with a similar viral genome copy number, but by 14 dpi, they exhibit a transient rise in viral genome copy number not seen in WT mice suggestive of HSV-1 reactivation from latency. A similar transient rise in copy number is also seen in WT mice depleted of CD8 T cells. Further, neither endogenous CD8 T cells within Pfn KO nor exogenous HSV-CD8 from GrB KO latently infected TG maintain latency as efficiently as CD8 T cells from WT TG in ex vivo TG cultures. Based on the current lack of evidence that CD8 T cell protection leads to neuronal death, we hypothesize a non-lethal LG-mediated block in HSV-1 reactivation from latency and are currently investigating possible mechanisms of protection.
NKG2D Regulates Viral Load in the CNS During Acute Picornavirus Infection (43.37)
Authors: Deb Chandra , Howe Charles
Published: January 2023

Neurovirulent picornaviral infections are a significant health risk in developing nations. Unfortunately, the mechanisms of picornaviral clearance from the central nervous system (CNS) are less understood and therapeutic approaches are limited. We have established a mouse model of neurovirulent picornavirus infection using the Theiler’s murine encephalomyelitis virus (TMEV) which replicates during the first 12 days postinfection (dpi) primarily in the hippocampus but is cleared from the brain by 21 dpi. Intracerebral infection of TMEV triggers the early infiltration of natural killer cells (NKCs) followed by CD8+ T cells (CTLs) into the brain. We observed that both brain-infiltrating NKCs and CTLs express the NKG2D activating receptor on their surface and 50% of the the CTLs specific to the VP2121–130 viral capsid peptide also expressed NKG2D. Concomitantly, brains of infected mice expressed NKG2D ligands such as RAE-1, H-60 and MULT-1 and microglia were the cellular locus of these ligands. Importantly, systemic treatment with the NKG2D function blocking antibody CX5 resulted in a significant increase in viral load in the brain. These results suggest that activated and infected microglia stimulate NKCs and CTLs via NKG2D in a manner that restricts viral replication. NKG2D may thus serve as a potential therapeutic target to enhance viral clearance responses during CNS infection.

This work was supported by a grant from the National MS Society and by Donald and Frances Herdrich.
Non-infected, but not infected dendritic cells undergo maturation in vivo following vaccinia virus infection (43.42)
Authors: Yammani Rama D. , Alexander-Miller Martha A.
Published: January 2023

Recently there has been renewed interest in poxvirus pathogenesis. Vaccinia virus (VACV) serves as a useful model for the study of poxvirus infection in vivo. Understanding how vaccinia virus generates immunity necessitates an appreciation for how this virus interacts with dendritic cells (DC), which are the most potent activators of naïve T cells. A number of in vitro studies have shown that DC infected with VACV fail to undergo maturation. However, the effect of VACV infection on DC in vivo remains less defined. We have found that following intravenous administration of vaccinia virus, both CD8+ and CD8− dendritic cells are infected. The number of infected DC peaks at 6 hours and is highly decreased by 24h postinfection. Dendritic cells infected with vaccinia virus have an immature phenotype. However, non-infected dendritic cells, both CD8+ and CD8− subsets, show increased expression of CD80, CD86, and CD40, suggesting that there is a generalized maturation of non-infected DC. In contrast to the generalized upregulation of costimulatory molecules, cytokine analysis suggests that only a subset of these mature DC may be relevant for activation of T cells, as IL-12 production is restricted to a small percentage of cells. These findings provide new insights into the control of DC maturation in vivo following vaccinia virus infection.

This work was supported by NIH grant P01 AI60642.
PD-1 expression on memory CD8 and CD4 T-cell subsets in healthy humans (43.43)
Authors: Duraiswamy Jaikumar , Miller Joseph D , Masopust David , Ibegbu Chris C , Wu Hong , Freeman Gordon J , Ahmed Rafi
Published: January 2023

It has been shown that the PD-1 (programmed death-1) inhibitory pathway is an important regulator of virus-specific CD8 T cell immune responses during a chronic viral infection in mice. Recent reports in HIV-infected people have shown that PD-1 is significantly upregulated on HIV-specific CD8 T cells and these exhausted cells can be reinvigorated by PD-1 blockade. We have tested the profile of PD-1 expression on chronic virus-specific CD8 T cells in normal healthy virus carriers. Analyses of CD8 T-cell subsets revealed that PD-1 was preferentially expressed by effector memory T cells rather than naive or central memory T cells. PD-1 was expressed by most CD8 T cells specific for human chronic infections, such as Epstein-Barr virus and cytomegalovirus, and at a lower level among CD8 T cells specific for infections that are cleared, such as influenza and vaccinia viruses. Blockade of PD-1 signaling using anti-PD-L1 antibody enhanced the ability of virus specific PD-1+ CD8 T cells to proliferate in response to antigen. These data support a role for PD-1 in suppressing CD8 T cells during chronic viral infections in humans.

The first two authors contributed equally to this work.
Interaction of Respiratory Dendritic cells with Influenza Virus Infection in vitro (43.44)
Authors: Hao Xueli , Braciale Thomas
Published: January 2023

Influenza A virus is a major cause of infection of the respiratory tract worldwide. Approximately 10–20% of U.S. population is estimated to be infected by this virus each year. Respiratory tract dendritic cells (RDC) are believed to play a major role as antigen presenting cells in initiating adaptive immune response to influenza virus infection. We have examined the effect of influenza virus infection of purified RDC in vitro at different MOI (Multiplicity of Infection) on virus protein expression, co-stimulatory molecules expression, and cytokines production. We found that there is not only an MOI dependent increase in the percentage of infected RDC, but also the infection of RDC at high MOI results in higher level and accelerated viral protein synthesis at early time post infection. Production of tested cytokines and chemokines by RDC increases with increasing of MOI except for IL-12 p40. Furthermore, we found that at high MOI total RDCs can be divided to several populations based on CD40 and influenza nucleoprotein (NP) expression: CD40high NPlow, CD40low NPhigh, and CD40negNPnegDCs. In conclusion, our results suggest that there is a diverse response of RDC to influenza virus at different MOI as reflected by their susceptibility to infection and cytokine production. These results may provide insight into how RDC modulate host response in the lung to influenza virus infection.

(Support: USPHS grant, NIH AI-15608, HL-33391,HL-71875)
The Characterization of Dendritic Cells in the MAIDS Model (43.46)
Authors: Giles Josephine Ruth , Nardiello Tricia Lynn , Stranford Sharon
Published: January 2023

Murine acquired immunodeficiency syndrome (MAIDS) is an animal model used to study human AIDS and is induced by murine leukemia virus. There are two different strains of mice used in this model system; BALB/c animals are naturally resistant and C57BL/6 are susceptible to MAIDS. Our laboratory’s previous studies examined differential immune responses during the first week of infection by comparing lymphoid organ gene expression patterns in BALB/c and C57BL/6 mice using DNA microarrays. Based on these data, it appears that the inability of C57/BL6 mice to develop an effective immune response may be due, in part, to changes in dendritic cell function or homing to lymph nodes. Based on microarray results, we hypothesize that this is due to lymphatic vessel remodeling differences. In the first part of this study, strain-specific differences in the mRNA expression patterns of specific DC function related genes identified by DNA microarray (e.g., CD83, DC-SIGN, and DCAR) were quantified using real-time RT-PCR.

In the second part of this study, immunofluorescent staining was used to visualize and quantify differences in the lymphatic vessel networks in the lymph nodes of disease resistant versus susceptible mice after MuLV infection. Our data suggest that by 3 days post infection, lymphatic vessel branching is more extensive in BALB/c mice (MAIDS resistant) than C57BL/6 animals (MAIDS susceptible). Further studies are underway to support and further quantify these preliminary results.
Early Events in the T cell Response to Respiratory Virus Infection - ‘Conveyor belt’ like sequential activation of CD8+ T cells in the lymph nodes. (43.48)
Authors: Yoon Heesik , Legge Kevin L , Sung Sun-Sang J , Braciale Thomas J
Published: January 2023

We have examined the tempo of T cell activation and proliferation in the draining lymph nodes in response to type A influenza virus infections with TCR transgenic CD8+ T cells. The CD8+ T cell activation/proliferation revealed by CFSE intensity correlated with the tempo of accelerated respiratory dendritic cells (RDC) migration from the infected lungs to the draining lymph nodes in response to influenza virus infection. In vivo administration of blocking anti-CD62L antibody at various time points before and after infection revealed that the virus specific CD8+ T cells entered the lymph nodes and activated in a sequential ‘conveyor belt’ like fashion. In conclusion, the onset of CD8+ T cell activation/proliferation after viral infection is controlled by the availability of DC harboring influenza antigens and the migration of these DC from the lungs to the draining lymph nodes.

This work is supported by USPHS grants AIO15608, HL03391, and HL071875.
Unique features of Vγ2Vδ2 T cell memory provides for bridge immunity to infections (44.16)
Authors: Morita Craig T. , Puan Kia-Joo , Lee Hoi K. , Wang Hong , Raker Amy M. , Moser Bernhard , Kuzuyama Tomohisa , Jin Chenggang
Published: January 2023

Human Vγ2Vδ2 T cells play important roles in mediating immunity against microbial pathogens and have potent anti-tumor activity. They recognize prenyl pyrophosphates such as isopentenyl pyrophosphate (IPP). We and others find that hydroxymethyl-butenyl pyrophosphate (HMBPP) is the major Vγ2Vδ2 antigen for many eubacteria. HMBPP is an intermediate in the MEP pathway for isoprenoid biosynthesis used by bacteria and protozoans. HMBPP was a 30,000-fold more potent antigen than IPP. The ubiquitous presence of prenyl pyrophosphates results in unique features of Vγ2Vδ2 T cell memory. Although exhibiting similar kinetics of expansion as CD8 αβ T cells after infections, almost all adult γδ T cells are memory cells that can be distinguished by expression of CD28, CD27 and CD45RA. High proportions of intermediate/late effector memory cells can be found in some donors even in early adulthood. Each memory subset displays distinct functional properties and chemokine receptors. Intermediate/late memory cells express CXCR1, CXCR2 and CX3CR1, whereas early central memory cells express CXCR6, CCR1 and CCR2. Late effector cells proliferate poorly compared with early and intermediate cells. Thus, different inflammatory chemokines attract distinct Vγ2Vδ2 memory subsets with different functional capabilities. The rapid conversion from naïve to memory T cells due to the recognition of a widespread microbial metabolite allows Vγ2Vδ2 T cells to mount memory responses to primary infections by bacteria and parasites using the MEP pathway.
Protective role of CCL6 during severe sepsis through interferon-γ generation and interferon-producing killer dendritic cells recruitment (44.18)
Authors: Coelho Ana Lucia , Schaller Matthew , Hogaboam Cory , Kunkel Steven
Published: January 2023

Our group has previously demonstrated that CCL6 enhanced antimicrobial immunity during sepsis through an unknown mechanism. The goal of this study was to investigate the mechanism involved in the protective role of CCL6 during the severe sepsis. C57BL/6 transgenically overexpressing CCL6 (Tg) in the lung and wild type (WT) mice were submitted to cecal ligation and puncture (CLP) and sham operation, and survival was evaluated for up to 7 days post-CLP. Cells from peritoneal cavity (PC) were obtained 3 days after surgery. The CCL6 Tg group exhibited greater levels of CCL6 in the lung and PC when compared to WT group after sham and CLP surgery. While CCL6 Tg mice presented 90% of survival at day 7 only 20% of WT mice survived. High levels of interferon-γ (IFN-γ) were detected in the PC of CCL6 Tg mice at 3 days post-CLP. Also, cells obtained from PC of these mice produced significantly levels of IFN-γ. When CCL6 Tg mice received anti-IFN-γ prior CLP, only 20% were alive. At 3 days after sham and CLP operation, the number of natural killer (NK) cells and interferon-producing killer dendritic cells (IKDCs) was increased in the peritoneal cavity of CCL6 Tg mice when compared to WT mice. These data suggest that the protective role of CCL6 may be due to its ability to contain the consequences of an inappropriate type 2-cytokine response during anti-pathogen responses. CCL6 may achieve this through promotion of IFN-γ and NK cells and IKDCs recruitment.
ATP-binding to the CATERPILLER protein Monarch-1, is required for its inhibitory function (44.19)
Authors: Ye Zhengmao , Lich John D , Moore Christopher , Williams Kristi L , Duncan Joseph A , Ting Jenny P._Y
Published: January 2023

The newly discovered CATERPILLER (NOD, NLR) family is defined by a nucleotide-binding domain (NBD) that consists of the Walker A and B motifs and ten additional motifs. It is conserved from plants to humans, and is important in immune regulation and several genetic disorders. Although the NBD is a defining feature of this family, there is a paucity of data to indicate that these proteins bind nucleotides, or the binding of nucleotides modulate their functions. Monarch-1/Pypaf7 is a negative regulator of IRAK-1 and NIK. In this report, we successfully purified recombinant Monarch-1 proteins to homogeneity. Purified Monarch-1 binds ATP but not CTP, GTP and UTP and exhibits ATP hydrolysis activity. Intact Walker A/B sequences are required for nucleotide binding. Overexpression of Monarch-1 nucleotide-binding defective mutant in THP-1 cells resulted in a dramatic increase of IL-6, chemokine CCL1, CXCL6 and CXCL13 to the extent that is comparable to the THP-1 Monarch-1 shRNA knock down cells in response to TLR2 agonist. This indicates that ATP binding is required for the inhibitory function of Monarch-1 and the nucleotide binding-defective mutant displays a dominant negative effect. Intact nucleotide-binding domain is also required for IRAK-1 dephosphorylation and p100 processing suggesting a nucleotide binding-dependent mechanism of Monarch-1 function in both canonical and non-canonical NF-κB pathway.
Antisense gene therapy in thermally injured mice orally infected with Enterococcus faecalis (44.27)
Authors: Shigematsu Kenji , Yoshida Tsuyoshi , Tsuda Yasuhiro , Kobayashi Makiko , Herndon David N , Suzuki Fujio
Published: January 2023

Infectious complications stemming from E. faecalis translocation in burn mice are influenced by a special subset of neutrophils (immunosuppressive PMN, PMN-II). CCL2 released from PMN-II has been characterized as an effector molecule when translocated bacteria spread systemically in these mice. In the present study, we tried to protect burn mice orally infected with E. faecalis using antisense gene therapy. c-fos antisense ODN was utilized in this gene therapy, because c-fos was described as a promoter of CCL2 gene expression. One day after burn injury (3rd degree, 25% TBSA burn), decontaminated BALB/c mice were infected orally with a lethal dose of E. faecalis (107 CFU/mouse), and treated s.c. with c-fos antisense ODN 6 hrs before, and 2 and 4 days after infection. These mice were observed once daily for a week, to determine their survival rates. In some experiments, sera obtained from burn mice, that were treated with or without c-fos antisense ODN, were assayed for CCL2 using ELISA. In the results, after E. faecalis oral infection, 90% of burn mice subjected to antisense therapy survived, while an 80% mortality rate was shown in the same mice treated with c-fos sense ODN. CCL2 was not detected in sera of burn mice treated with c-fos antisense ODN. These results indicate that infectious complications caused by E. faecalis translocation are controllable in thermally injured mice by antisense gene therapy using c-fos antisense ODN.

This study was supported by SHC NA #8840.
Regulation of neutrophil-mediated host defense to pneumococcal infection by NKT and gamma-delta T cells (44.34)
Authors: Hatta Masumitsu , Nakamatsu Masashi , Nakasone Chikara , Fujita Jiro , Kaku Mitsuo , Kawakami Kazuyoshi
Published: January 2023

Recently, NKT and gamma-delta T cells have garnered attention in the host defense to various infectious pathogens. In the current study, we examined the role of these lymphocytes in neutrophil-mediated host defense to pulmonary infection with Streptococcus pneumoniae, a leading causative bacterium in community-acquired pneumonia. Both J alpha 18 KO and V gamma 4 KO mice, lacking iNKT cells and TCR-V gamma 4+ T cells respectively, were more susceptible to this infection than wild-type (WT) mice, which was associated with attenuated synthesis of TNF-alpha and MIP-2 and reduced accumulation of neutrophils in lung. The attenuated responses and host defense in J alpha 18 KO mice were completely recovered by administration of either rIFN-gamma or liver mononuclear cells (LMNC) obtained from WT mice, which effect was abrogated when LMNC were prepared from IFN-gamma KO mice. By contrast, such recovery effect by rIFN-gamma administration was not or marginally observed in V gamma 4 KO mice. Taken together, these results demonstrate that both iNKT cells and gamma-delta T cells play an important role in neutrophil-mediated host defense to pneumococcal infection and suggest that these lymphocytes may work in a different mechanism.
IFN-λ contributes to dendritic cell-mediated expansion of regulatory T cells during HCV infection. (44.38)
Authors: Dolganiuc Angela , Paek Edward , Fair Joshlean , Szabo Gyongyi
Published: January 2023

IFN-λ inhibits HCV replication and triggers IFN-dependent genes. We hypothesized that IFN-λ is immunomodulatory and evaluated its effects on myeloid dendritic cells (DC). DCs generated with IFN-λ (IFN-λ-DC) expressed normal levels of DC markers, but showed reduced allostimulatory capacity in mixed lymphocyte reaction (MLR), similar to DCs of patients with chronic HCV infection (HCV-DC). No additive effects of IFN-λ subtypes were observed. The inhibition of MLR was due to preferential expansion of CD4+CD25+ T cells in the presence of HCV-DCs or IFN-λ-DC, but not normal DCs. Purified CD4+CD25+ T cells from MLR were Foxp3+, CD45RA+, produced IL-10 and TGFb, and inhibited CD4+CD25− T cell proliferation. HCV-DCs produced increased levels of IFN-λ compared to controls. In vitro generation of DCs in the presence of LPS increased expression of IFN-λ. Interestingly, we found increased plasma levels of LPS in HCV patients compared to controls suggesting a possible in vivo mechanism for IFN-λ induction. Further, in vitro DCs generation with LPS resulted in expansion of Tregs and inhibition of MLR, thus mimicking the effects of HCV-DCs and IFN-λ-DCs. We also identified an increased frequency of Tregs in peripheral blood of HCV patients (12±2%) compared to controls (5±1%) (p<0.05). In conclusion, increased frequency of Tregs in HCV infection is associated with increased IFN-λ production by DCs that promote Treg generation.
MyD88-mediated signals induce in vivo production of the bactericidal lectin RegIIIγ and protect against intestinal Listeria monocytogenes infection (44.4)
Authors: Brandl Katharina , Plitas George , DeMatteo Ronald P. , Hooper Laura V. , Pamer Eric G.
Published: January 2023

Listeria monocytogenes (L. monocytogenes) is an intracellular bacterium that causes systemic infections after traversing the intestinal mucosa. To test the hypothesis that the Toll-like receptor pathway is involved in defense against intestinal L. monocytogenes infection, we analyzed the role of the common intracellular adaptor molecule myeloid differentiation primary-response protein 88 (MyD88) following oral infection with L. monocytogenes. We found that MyD88 deficient mice have increased susceptibility to intestinal L. monocytogenes infection with higher bacterial burden in spleen, liver, mesenteric lymph nodes, intestinal lumen, and small intestine (lamina propria and intestinal epithelium). Further in vitro and in vivo studies showed that MyD88 mediated protection primarily occurs in the distal small intestine. Recently, it was shown that the intestinal bactericidal lectin RegIIIγ has antibacterial activity against L. monocytogenes. RT-PCR and Western Blot analysis of distal small intestines showed that RegIIIγ is significantly diminished in MyD88 deficient mice. Since optimal expression of RegIIIγ requires MyD88-mediated signals, our findings suggest that increased susceptibility to intestinal L. monocytogenes infection in MyD88-deficient mice results from diminished levels of this bactericidal lectin in the distal small bowel.
Characterization of constitutive antimicrobial activity associated with leukocyte lysates from the nurse shark, Ginglymostoma cirratum (44.42)
Authors: Hinds Nichole A , Smith Sylvia L
Published: January 2023

Antimicrobial peptides are mediators of innate immunity. Studies have shown that peptide fragments of histones, proteins primarily involved with DNA packaging, display antimicrobial activity. This project examines the antibacterial activity associated with shark leukocyte lysates and the potential role of histone fragments in constitutive innate immunity. Preliminary studies have demonstrated antimicrobial activity, distinct from that of lysozyme, in crude cell lysates prepared from unstimulated shark blood leukocytes. The antimicrobial activity has been partially purified using a combination of ion exchange, size exclusion and affinity chromatography. SDS PAGE analysis of fractions showed the presence of several low molecular weight (<15 kDa) protein bands. N-terminal amino acid sequence analysis of four peptides identified them as histone fragments with sequence similarity to mammalian histones H2A, H2B and H4. The peptide sequences are also similar to histone fragments described for the catfish and the Asian toad, which exhibit antibacterial activity. The spectrum of antibacterial activity, biochemical characterization and mode of action of shark histone fragments is under investigation. Such information will determine their potential use against clinically significant organisms.

The research was supported in part by FIU’s Comparative Immunology Initiative (NIH R25GM061347).
HIV-1 induces targeted down-regulation of the Ig gene-diversifying enzyme AID in the germinal center of infected lymphoid follicles (45.1)
Authors: Santini Paul A , He Bing , Chiu April , Ball Susan C , Chen Kang , Kingsley Lawrence A , Rinaldo Charles R , Knowles Daniel M , Chadburn Amy , Xu Weifeng , Cerutti Andrea
Published: January 2023

Class switching from IgM to IgG and IgA is essential for antiviral immunity and requires activation-induced cytidine deaminase (AID), an APOBEC family member with DNA-editing activity. Germinal center (GC) B cells express AID upon activation by CD4+ T cells through CD40 ligand (CD40L) and IL-4. HIV-1 is thought to impair IgG and IgA responses to viral antigens, opportunistic pathogens and vaccines by causing progressive loss of CD4+ T cells and by rendering B cells poorly responsive to CD4+ T cell help. It remains unknown whether HIV-1 targets AID to hamper protective IgG and IgA responses. We found that infected GCs contained less AID, but normal APOBEC3G, an AID-related RNA-editing protein. AID down-regulation was not associated with local loss of CD4+ T cells and CD40L, but rather correlated with decreased activation of AID-inducing transcription factors, such as NF-κB and STAT6, and with increased expression of feedback inhibitors of NF-κB and STAT6, including IκBα, SOCS1 and SOCS3. AID down-regulation also correlated with accumulation of the viral protein Nef in the GC and with trafficking of Nef within membrane channels connecting infected myeloid cells to B cells. Together with our recent in vitro studies showing that Nef penetrates B cells and inhibits class, the present in vivo data suggest that HIV-1 evades protective IgG and IgA responses by targeting the class switch recombinase machinery.
Differential Protease Activity in the Secondary Lymphoid Tissues of Disease Resistant and Susceptible Mouse Strains in the MAIDS Model (45.12)
Authors: Nardiello Tricia Lynn , Giles Josephine Ruth , Stranford Sharon A.
Published: January 2023

Murine leukemia virus (MuLV) is used as a mouse model system to study human AIDS. There are two strains of mice used in this model, BALB/c and C57 BL/6. Although both strains of mice will become infected, BALB/c strain recover while the BL/6 strain becomes immunocompromised (MAIDS). Previous DNA microarray work comparing the two strains has shown that certain proteases are up regulated in the BALB/c lymph nodes and spleen shortly after infection. Lymphatic remolding and cell activation are possible consequences of protease activity, and therefore the up regulation of these proteases in the BALB/c strain may be important for recovery.

Using immunofluoresent microscopy and in situ zymography, quantification of protease activity in the lymph nodes of each strain was studied in the first days after MuLV infection. Serial sections were stained for lymphatic (LYVE-1) and vascular (CD31) endothelial structures using primary and fluorescently-conjugated secondary antibodies. This was combined with protease-mediated activation of quenched substrate fluorphores (in situ zymography) allowing co-localization of protease activity and lymphatic morphology.

Preliminary in situ zymography results show that there is an increase in protease activity in BALB/c mice when compared to BL/6 in the lymph nodes. Furthermore, preliminary immunofluorescent results show an increase in the vascular and lymphatic branching within the BALB/c lymph nodes following MuLV infection.
Characterization of the kinetics and magnitude of the immune response to rhesus rhadinovirus in immune competent and immune deficient macaques (45.14)
Authors: Messaoudi Ilhem , Powers Michael F. , Wong Scott W
Published: January 2023

Rhesus Rhadinovirus (RRV) is closely related to human herpesvirus 8 (HHV-8) or Kaposi’s sarcoma-associated herpesvirus (KSHV). In rhesus macaques (RM) previously inoculated with simian immunodeficiency virus (SIV), RRV infection results in the development of several B cell lymphoproliferative disorders similar to those seen in AIDS patients infected with KSHV. In this study we compared the adaptive immune response to RRV infection generated in both immune deficient (SIV+) and immune competent (SIV-) RM. We evaluated the kinetics and size of the proliferative burst to RRV infection by determining the prevalence of Ki-67+ cells within CD4, CD8b, and CD20 positive subsets. Furthermore, we measured the prevalence of antigen specific T cells in peripheral blood as well as bronchial alveolar lavage. Our data indicates that immune competent animals generate substantially larger CD8 and CD20 responses, albeit with similar kinetics, than immune deficient animals. Furthermore, immune deficient animals show higher viral loads and sustained viremia, suggesting that CD8 and CD20 responses are critical in the initial control of RRV viral growth and spread. Interestingly, antigen specific responses are more easily detected in peripheral blood of immune deficient animals.
Stress-Induced Changes In Immune Cell Populations In Mice Exposed To Hindlimb Unloading Restraint (45.19)
Authors: O’Donnell Phyllis M , Aviles Hernan O , Orshal Julia M , Sonnenfeld Gerald
Published: January 2023

Hindlimb unloading (HU) is a rodent model that has been successfully used to simulate some of the aspects of space flight including alterations of the immune system. We have shown in previous studies that exposure of mice to HU results in significantly increased plasma norepinephrine levels, decreased IL-6 and TNF-α from LPS stimulated spleen cell supernatants, and increased susceptibility to bacterial infection compared to control mice. The purpose of this study was to examine the populations of immune cells affected by HU to better understand the mechanisms by which HU induced changes in susceptibility to infection. Spleens from HU or control normally caged mice were asceptically removed after 18 or 48 hours of HU exposure, weighed, and analyzed for cell antigen marker expression. Spleen weights were significantly reduced in the HU group with no significant changes in body weights. Flow cytometric analyses revealed that the percentage of splenic CD19+ B cells in mice exposed to HU was significantly reduced at both 18 and 48 hr following HU commencement compared to controls. CD4:CD8 ratios declined from control 2.3 to 2.2 with 18 hr HU and to 1.9 with 48 hr HU. These results suggest that lymphocyte sub-population changes may contribute to the dysregulation of immunity in mice exposed to HU and the subsequent reduced ability of these mice to resist infections.

Funded in part by a grant from the Amino Up Chemical Company (Sapporo, Japan).
MECHANISMS OF NEUTROPHIL DEATH IN HIV INFECTED PATIENTS: ROLE OF ROS, CASPASES AND MAP KINASES PATHWAYS (46.14)
Authors: Salmen Siham , Montes Henry , Soyano Andres , Hernandez Dimas , Berrueta Lisbeth
Published: January 2023

Neutrophils from HIV+ patients have an increased susceptibility to undergo programmed cell death (PCD), which could explain neutropenia during advanced disease. In this work, key steps of PCD have been evaluated in neutrophils from HIV+ patients. The role of caspase-3, caspase-8, MAPK and ROS, were analyzed. Spontaneous neutrophil death is caspase-3 but no caspase-8 dependent, suggesting that the intrinsic pathway is involved as a pathogenic mechanism of PCD. Inhibition of ROS decreased spontaneous PCD and caspase-3 hydrolysis, connecting oxidative stress and Caspase-3 within neutrophils PCD in HIV infected patients. An increased constitutive phosphorylation of p38 MAPK, associated to increased neutrophils death in HIV+ patients, following inhibition of this kinase, was observed, suggesting a role for p38 MAPK in cell survival during the disease. We conclude that oxidative stress secondary to HIV infection can accelerate neutrophil death.
CD103-expressing CD4+Foxp3+ regulatory T cells are critical for the survival of mice infected with the helminth parasite Schistosoma mansoni. (46.17)
Authors: Hesse Matthias , Wilson Mark S. , Cheever Allen W. , Wynn Thomas A. , Bajracharya Siddhartha
Published: January 2023

The complicated life cycle of helminths requires a lasting infection of their hosts. The parasites must avoid destruction by the host’s immune response but also prevent exacerbated immunopathology. Activation of immunoregulatory mechanisms during helminth infection establishes a protective homeostatic immune response. We reported the presence of CD4+Foxp3+regulatory T cells (naTreg) in S. mansoni-egg-induced hepatic granulomas. In immunizations they controlled egg-induced cytokine production. Strikingly, with disease progression the frequency of CD103+naTregs significantly increased. CD103 (the αE chain of αEβ7 integrin) is an activation marker for naTregs with potential roles for migration/maintenance of naTregs at inflammatory sites. Using CD103-deficient mice we investigated the role of CD103 in schistosomiasis. Most infected CD103-KO mice succumbed during transition from acute to chronic inflammation. They developed lower Th-2 responses, reduced Th-2-pathology but exacerbated liver damage. The frequency of naTregs in granulomas was not changed. Importantly, transfer of CD103-competent naTregs rescued infected CD103-KO mice. We demonstrate for the first time the essential protective role of naTregs in schistosome-infected immunocompetent hosts. Although CD103 is not necessary for the presence of naTregs in granulomas, it seems to be pivotal for their function in schistosomiasis.