bluuebunny/crossref_metadata_2025 huggingface dataset:
The chemokine receptor CXCR3 is required for optimal murine resistance against Toxoplasma gondii (129.30)
Authors: Snider Heidi M , Lockhart Gabriel , Barbi Joseph , Papenfuss Tracey L , Lu Bao , Satoskar Abhay R
Published: January 2023

CXC chemokine receptor 3 (CXCR3) plays a critical role in recruitment of neutrophils, natural killer cells and T cells to sites of inflammation. Since these cell types are all critical in control of Toxoplasma gondii infection we hypothesized that CXCR3 is critical for immunity against T. gondii. To test this hypothesis we compared the course of T. gondii (ME49) infection in WT and CXCR3-/- mice on a C57BL/6 background. Following ME49 challenge, CXCR3-/- mice developed high brain parasite loads and succumbed to infection significantly faster than WT mice. T. gondii antigen-stimulated splenocytes from CXCR3-/- mice produced significantly more IFN-γ than WT splenocytes, but serum levels of this cytokine were lower in the former. Flow cytometry also revealed that CXCR3-/- mice recruit fewer leukocytes into the brain than WT mice early during infection. Finally, adoptive transfer studies involving Rag2-/- mice reconstituted with CXCR3-/- or WT lymphocytes revealed that CXCR3 expression on T cells was critical for effective immunity against ME49. Taken together, these results show that CXCR3 is critical for immunity against T. gondii. Furthermore, they suggest that increased susceptibility of CXCR3-/- mice is due to impaired leukocyte trafficking - most likely CXCR3 expressing T cells - into infection sites and not due to impairments in type I CD4+ T cell responses.

Support: NIH
Functional Consequences of Increased Virus-specific CD8+ Effector Memory T cells in the Response to Influenza in Older Adults (130.4)
Authors: Zhou Xin , McElhaney Janet E
Published: January 2023

Background: The decline in adaptive immune function with age is a major cause for increased risk of complicated influenza illness in older adults especially those with congestive heart failure (CHF). Changes in the CD8+ T cell response to the virus may explain the increased risk of influenza in the older population. Methods: The response to influenza in 12-hour peripheral blood mononuclear cell (PBMC) cultures from vaccinated healthy young (20-40 y.o.) and older adults (age =60 y.o.) with or without CHF was measured in different CD8+ T cell subsets. Results: In unstimulated PBMC, older compared to young adults showed a higher proportion of GrB+CD8+ T cells, and these T cells also expressed higher levels of CD107a in the CHF group. Healthy young and older adults showed a significant increase in the GrzB+CD8+ T cells expressing CD107a, consistent with a cytolytic effector function; older adults with CHF had no response. Preliminary results suggest that this poor response is associated with an increased proportion of terminally-differentiated, effector memory (CD8+CD45RA+) T cells. Conclusions: An accumulation of terminally differentiated CD8+CD45RA+ T cells have an effector phenotype but do not respond to influenza virus and may explain the increased risk of influenza in the older adults.
Macrophages contribute to pathogenesis of visceral leishmaniasis via a STAT1-dependent mechanism (131.10)
Authors: Snider Heidi M , Barbi Joseph , Satoskar Abhay R
Published: January 2023

We previously reported that STAT1-/- mice on a susceptible BALB/c background were highly resistant to visceral leishmaniasis (VL) and developed minimal organ pathology compared with wild-type (WT) controls, despite mounting a poor Th1 response. We also found that STAT1 in non-T cells (i.e. macrophages and neutrophils) mediated susceptibility and pathology. The objective of this study was to determine whether macrophages are involved in the pathogenesis of VL cause by Leishmania donovani and to determine the role of STAT1 in phagocytes in mediating disease susceptibility. Depletion of monocytes and macrophages using clodronate-containing liposomes both prior to and after L. donovani infection impaired Th1 development in WT BALB/c mice, but rendered them highly resistant to infection and minimized liver pathology. This phenotype was strikingly similar to that seen in infected STAT1-/- mice. Further in vivo studies using an airpouch model demonstrated that both macrophages and neutrophils of STAT1-/- mice had significant impairments in parasite uptake compared with WT controls. Taken together, these results demonstrate that macrophages are involved in the pathogenesis of VL, and that STAT1 signaling may mediate disease susceptibility by facilitating parasite uptake into host phagocytes.

Support: NIH
Molecular insights into how the adenovirus E3-19K protein associates with MHC class I molecules (131.5)
Authors: Fu Jie , Bouvier Marlene
Published: January 2023

The immunomodulatory protein E3-19K from Adenoviruses (Ads) blocks cell surface expression of MHC class I by preventing their export from endoplasmic reticulum (ER), thus evading host's antiviral cellular immune responses. Currently, structural information on E3-19K and the biochemical properties of the E3-19K/class I complex is lacking. This knowledge will help not only to better understand host/virus interaction, but also to design effective antiviral therapies against Ad infections. Using a proteolytic approach, we showed that the recombinant, soluble E3-19K (residues 1-100) generated a large N- (residues 1-88) and small C- (residues 94-100) terminal fragments which encompass the majority of residues within the variable and conserved regions, respectively, of E3-19K. We showed that fragments corresponding to the variable and conserved regions do not remain associated together after proteolysis. Moreover, neither of these fragments associates with class I. These results strongly suggest that the ability of E3-19K to bind to class I molecules depends on a delicate structural interplay between the variable and conserved regions. We also identified residue 93 in E3-19K as critical for E3-19K association with class I molecules. Together, these results provide novel insights into the structure/function relationship of E3-19K and its effect on down regulation of class I molecules. Funded by NIH AI65943.
Enhanced NKT cell response and skewed polarization of DC in RSV infection and susceptibility to asthma (131.6)
Authors: Sun Jonathan , Durre Kainath , Qureshi Mahboob
Published: January 2023

RSV infection in early infancy makes individuals susceptible to develop asthma in later life due to airway hyper-responsiveness (AHR) to innocuous antigens, which is characterized by enhanced Th2 responses. Using a neonatal mouse model, we investigated whether differential NKT cell responses and skewed polarization of DC are involved in the underlying mechanism. Flow cytometry revealed a significant increase in the proportions of NKT cells (CD3-CD19-CD1d+) in RSV-infected 5 days old lungs compared to that of 7 and 34 days old lungs. A comparable increase in the proportions of plasmacytoid DCs (pDCs; CD11blo CD11clo CD45R+) to myeloid DC (mDCs; CD11blo CD11chi/med CD45R-) was also determined by flow cytometry. To note, DC phenotypes involved in antigen presentation influence the outcomes of the immune responses. In support of this notion, RSV infection induced a significantly higher production of Th2 cytokines than Th1 cytokines in the lungs of younglings. In contrast, RSV-infected adult lungs produced significantly higher amount of Th1 cytokines. Furthermore, NK cell response was not enhanced in the young lungs as opposed to that in the adult lungs in response to RSV infection. Thus, enhanced NKT cell and reduced NK cell responses to RSV infection in the neonatal lungs, along with an enhanced Th2 response, may have induced a skewed polarization of DCs to pDCs leading to development of AHR and susceptibility to asthma.
Destruction of monocyte/macrophages dictate AIDS progression in SIV infected macaques (131.9)
Authors: Kuroda Marcelo J. , Liu Huining , Ling Binhua , Sugimoto Chie , Williams Kenneth , Lackner Andrew A. , Veazey Ronald S. , Hasegawa Atsuhiko
Published: January 2023

It is widely accepted that destruction of CD4+ T cells and viral load are the primary markers for immunodeficiency in HIV-1-infected humans and in simian immunodeficiency virus (SIV)-infected macaques. However, the mechanisms that dictate the tempo of disease progression have yet to be elucidated. Macrophages, an important cell component of the innate immune system and link between innate and adaptive immunity, are also important targets of HIV/SIV infection. We therefore examined whether changes in cells of monocyte/macrophage lineage could be linked to the pathogenesis of AIDS in the rhesus macaque model. Here we show that massive turnover of peripheral monocytes associated with death of tissue macrophages correlates with AIDS progression in macaques. More importantly, the level of monocyte turnover was not linked to the CD4+ T cell count and was a better predictive marker for AIDS progression than viral load or lymphocyte activation. Our results show the importance of monocyte/macrophages in the pathogenesis of AIDS and suggest the dynamic changes of the monocyte/macrophages as a new marker for AIDS progression.
Priming of multifunctional Mycobacterium tuberculosis-specific CD8 T cells (132.13)
Authors: Einarsdottir Thorbjorg , Flynn JoAnne L.
Published: January 2023

M. tuberculosis (Mtb) infection in mice results in a slow but steady increase in bacterial numbers in the lungs, peaking at 3-4 weeks, followed by a state of chronic infection with bacterial numbers slightly lower than the peak. While T cells have been shown to be important for containing Mtb infection, the only known method of clearing the bacteria is by extended antibiotic treatment. Our previous results showed that Mtb-specific CD8 T cells were limited to either cytotoxicity or secretion of IFN-g, indicating a functional defect in the cells. The majority of Mtb-specific CD8 T cells were cytotoxic, with IFN-g production limited to early responses (at 3-4 weeks). In the current study, we used a recombinant Mtb strain expressing ovalbumin epitopes (Mtb-ova) to examine the functional profile of naïve ovalbumin-specific CD8 T cells adoptively transferred to mice at different times post-infection. Cells transferred before infection acquired primarily cytotoxic function, while cells transferred at 6 weeks became either cytotoxic or secreted IFN-g. Cells transferred at the peak of infection (at 3 weeks) were more multifunctional than cells transferred at other times, in that they could be both cytotoxic and secrete IFN-g. Future work will focus on identifying what determines the function of the cells and whether an increase in multifunctional cells provides improved bacterial control.

Funding: NIH/NIAID AI37859-10.
Anti-West Nile Virus antibodies provide protection from WNV infection: protective antibodies recognize diverse WNV proteins (132.21)
Authors: Konschak Sarah R , Petell Christopher , Schiltz James , Petell James , Bradley David S.
Published: January 2023

We demonstrate here that goose-derived anti-West Nile Virus (WNV) antibodies are both protective against WNV infection and specific for a broad spectrum of WNV proteins. WNV is a Flavivirus that predominantly infects birds via a mosquito vector. The majority of humans infected with WNV demonstrate mild flu-like symptoms. However, a small number of human WNV cases result in meningitis and encephalitis, which can ultimately lead to paralysis or death. The epidemiology of WNV infections indicates that a population-wide vaccine is neither necessary nor cost-effective. An effective passive therapeutic antibody treatment would, however, provide an excellent alternative for individuals with WNV infection involving the CNS. We have demonstrated that antibodies, purified from geese naturally infected with WNV, protects both avian and mammals from WNV infection. WNV-infected golden hamsters receiving goose anti-WNV antibodies demonstrated 100% survival while all of the infected animals receiving a sham treatment perished. Characterization of the WNV-specific goose antibodies indicates a polyclonal response with diverse specificities, including WNV proteins E, pre-M and NS3. This diverse reactivity suggests enhanced protection both in the recognition of various replicative and envelope components and potentially across various WNV strains.

DOE "Goose Diagnostic and Therapeutic Antibodies to West Nile Virus and AI"
JVRS-100 adjuvanted universal influenza A vaccine based on a multiple antigenic peptide of M2e protects from lethal challenge (132.6)
Authors: Callejo Bernadette J , Chang Stella , Lay Marla , Liang Lily , Fairman Jeffery
Published: January 2023

Background: The conventional vaccine strategy for control of influenza A is vulnerable to antigenic drift and the emergence of unmatched epidemic strains that cause primary vaccine failure. A vaccine strategy that targets an influenza antigen, which is less susceptible to antigenic variation, would be a major improvement. Methods: Balb/c mice were vaccinated three times with the M2e peptide in the context of a multiple antigenic peptide (MAP) complex and combination with the cationic lipid DNA complex adjuvant (JVRS-100). Antibody titers were monitored over the time course of vaccination and the mice received a lethal challenge with H1N1 (PR/8/34) or H3N2 (HKx31) two weeks after the final vaccination. Results: Vaccination of mice with JVRS-100-MAP4-M2e compared to MAP4-M2e resulted in increased survival following lethal challenge with 6x LD50 PR/8/34 H1N1 (100% vs. 30%, P=0.002) or 10x LD50 HKx31 H3N2 (80% vs. 20%). Recipients of 200µl serum from MAP-4-M2e/JVRS-100 vaccinated mice and challenged with H1N1 (6x LD50 PR/8/34) resulted in survival of 60% (P=0.014) compared with control mice which received 200µl naive serum (0% survival). Furthermore, recipients of 300µl serum from MAP-4-M2e/JVRS-100 vaccinated mice and challenged with H1N1 (2x LD50 PR/8/34) resulted in survival of 100% (P=0.0026) compared with control mice which received 200µl naive serum (0% survival). Conclusion: These studies demonstrate that a simple, synthetic M2e vaccine in a MAP configuration with a strong adjuvant may result in a viable vaccine candidate for universal flu vaccination and the protective immunity is primarily due to an antibody response.
Viruses Selectively Hide CD8 T Cell Epitopes. (132.7)
Authors: louzoun yoram , vider tal
Published: January 2023

Viruses employ various means to evade immune detection. One common evasion strategy is the removal of CD8 cytotoxic T-lymphocyte (CTL) epitopes. We here use a combination of multiple bioinformatic tools and large amount of genomic data to compute the epitope repertoire presented by over 1,300 viruses in multiple HLA alleles. We define the "Size of Immune Repertoire (SIR) score," which represents the ratio between the epitope density within a protein and the expected density. We show that virusal proteins in general have a higher epitope density than human proteins. This difference is due to a good fit to the human MHC system to the typical amino acid usage of viruses. Among different viruses, viruses infecting humans present less epitopes than non-human viruses. This selection is not at the amino acid usage level, but through the removal of specific epitopes. Within a single virus, not all the proteins express the same epitopes density. Proteins expressed early in the viral life cycle have a lower epitope density than late proteins. Such a difference is not observed in non-human viruses, The targeting of the CTL immune response to viral late structural and enzyme proteins, allow the virus time interval to propagate before its host cells are destroyed by CTLs.
Protective CD4 T cell-independent IgM responses impairs the production of isotype-switched antibodies during intracellular bacterial infection (133.14)
Authors: Racine Rachael , Chatterjee Madhumouli , Wittmer Susan , McLaughlin Maura , Jones Derek , MacNamara Katherine C , Winslow Gary
Published: January 2023

Ehrlichia muris, an intracellular tick-borne pathogen, generates a CD4 T cell independent (TI) protective response in C57BL/6 mice. At peak infection, the response is accompanied by the expansion splenic extrafollicular CD11clo-expressing plasmablasts that produce antigen-specific IgM. Using secretory IgM (sIgM)-deficient mice and activation-induced adenosine deaminase (AID)-deficient mice, which are unable to undergo antibody class-switching, we demonstrated that IgG is dispensable for immunity and unmutated IgM was sufficient for protection to challenge infection with a highly virulent ehrlichia. Furthermore, while IgM-secreting cells were abundant in the spleen, prior to day 21, IgG was largely absent and instead produced in infected LNs. Thus, the robust IgM response impaired the formation of germinal centers and production IgG-secreting cells in the spleen. These findings, in turn, suggested that ehrlichial infection might suppress the spleen's ability to produce isotype-switched antibodies to blood borne antigens or pathogens. Indeed, E. muris infected-mice immunized with NP-CGG exhibited an impaired NP-specific IgG response. Thus, our studies demonstrate a major protective role for CD4 T cell-independent IgM during intracellular bacterial infection, and suggest a mechanism whereby a T cell-independent response may suppress T-dependent responses during co-infecting pathogens.
Surfactant Protein D is cleaved by matrix metalloprotease-9, resulting in disruption of collectin function (133.16)
Authors: Weathington Nathaniel M , Jackson Patricia L , Wilson Landon , Gaggar Amit
Published: January 2023

Surfactant protein D (SP-D) is a pulmonary collectin, containing collagen and lectin domains, important in innate host defense in the lung. It agglutinates and opsonizes bacteria and leads to phagocytosis by macrophages. Matrix metalloproteinase-9 (MMP-9) is a gelatinase upregulated in many inflammatory diseases of the lung. We tested SP-D as a substrate for MMP-9, and assessed SP-D function in this context. We show cleavage at three discrete sites of the collagen domain, generating C-terminal fragments of 34, 27, and 19KDa identified by western blot and with specific cleavage sites verified by mass spectrometry. SP-D is not, however, hydrolyzed by the related protease MMP-8, nor the serine protease prolyl endopeptidase, both of which cleave collagen. Incubation with these three proteases separately or in combination did not generate any detectable quantity of the neutrophil chemoattractant proline-glycine-proline (PGP), though SP-D contains many PGP motifs in the collagen domain. Furthermore, with MMP-9 cleavage, SP-D's intrinsic neutrophil chemoattractant activity was eliminated and its ability to aggregate Pseudomonas aeruginosa was also decreased. These studies indicate that in inflammatory lung diseases where MMP-9 is upregulated, impairment of SP-D's innate immunologic properties may increase frequency and severity of pulmonary infection, emphasizing the role of MMP-9 in disease.
Extracellular bactericidal functions of porcine neutrophils (133.20)
Authors: Scapinello Sarah E. , Brooks Andrew S. , Caswell Jeff L. , Macinnes Janet I. , Hammermueller Jutta , Smith Alexandra K.
Published: January 2023

Neutrophils are one of the main effector cells of innate immunity and were shown to kill bacteria via phagocytosis more than 100 years ago. New developments, however, also show that neutrophils are capable of antimicrobial activity by producing extracellular structures named neutrophil extracellular traps (NETs). The antimicrobial effects of NETs can be attributed to cathelicidins, a category of antimicrobial peptides stored as inactive proforms. These proforms are cleaved by neutrophils elastase during cell activation in response to pathogens. This project focuses on the cathelicidin peptides contained within NETs and their effectiveness in killing several common bacterial pathogens of swine.

Subsequent investigations address the contribution of cathelicidins to extracellular bactericidal activity of porcine neutrophils. Activated secretions of neutrophils are effective in killing 100% of Escherichia coli K12, 21% of Streptococcus suis, and 74% of Actinobacillus suis. Pasteurella multocida appears to survive in the presence of activated neutrophil secretions demonstrating a 38% increase in growth when compared to P. multocida exposed to non activated neutrophils secretions. Identification of the contribution of cathelicidins to this bactericidal activity is pending. These data suggest that activated neutrophil secretions have bactericidal activity against common pathogens in swine.
Differences between pathogenic and non-pathogenic microsporidial-induced signaling in macrophages (133.30)
Authors: Mathews Amber , Fischer Jeff L , Hotard Annie , Donze Hollie Hale
Published: January 2023

Microsporidia are ubiquitous, obligate intracellular eukaryotes that cause chronic diarrhea in humans, especially in immunocompromised individuals. Little is known about the innate immune response to these parasites. Macrophages are a source of interleukin-12 (IL-12) and play an essential role in the link between innate and adaptive immunity. Previous studies from our lab have shown that pathogenic species, E. cuniculi and E. intestinalis, are preferentially recognized by TLR2 in primary human macrophages and produce IL-10 (Th2) responses. In this study, we are investigating the signaling cascades regulated by TLR2 and TLR4 engagement with pathogenic and non-pathogenic species of microsporidia. Because TLR4 engagement triggers MAPK activation leading to IL-12 production, we hypothesize that differences in TLR activation by non-pathogenic species will lead to activation of p38 MAPK and IL-12 production. Using small interfering RNA, p38 MAPK was knocked down in human macrophages and resulted in a decrease in IL-12p40 and tumor necrosis factor alpha (TNF-α) production when infected with Vairimorpha necatrix, a non-pathogenic species, and a decrease in TNF-α production when infected with E. cuniculi. Using western blot, we found that activated p38 MAPK is continuously expressed from 3h to 24h post infection of human macrophages with V. necatrix. Expression of activated p38 MAPK is transient when infected with E. cuniculi as we only detected activation at 3h and 6h post infection. These data suggests that activation of p38 MAPK is necessary for the appropriate innate immune response to microsporidia and for control of infection. This work is supported by NIH grant AIO71778.
Ly49H signaling through both DAP10 and DAP12 is required for optimal NK cell responses to mouse cytomegalovirus infection (133.39)
Authors: Orr Mark T , Lanier Lewis L
Published: January 2023

The activating natural killer (NK) cell receptor Ly49H recognizes the mouse cytomegalovirus (MCMV) m157 glycoprotein expressed on the surface of infected cells and is required for protection against MCMV. Although Ly49H has previously been shown to signal via DAP12, we now show that Ly49H must also associate with and signal via DAP10 for optimal function. In the absence of DAP12, DAP10 enables Ly49H-mediated killing of m157-bearing target cells, proliferation in response to MCMV infection, and partial protection against MCMV. DAP10-deficient Ly49H+ NK cells, expressing only Ly49H - DAP12 receptor complexes, are partially impaired in their ability to proliferate during MCMV infection, display an aberrant signaling profile, produce less IFN-γ upon Ly49H engagement, and demonstrate reduced control of MCMV infection. Deletion of both DAP10 and DAP12 completely abrogates Ly49H surface expression and control of MCMV infection. Thus, optimal NK cell-mediated immunity to MCMV depends on Ly49H signaling through both DAP10 and DAP12.
Peripheral blood monocytes (PBMCs) display innate antiviral response to rhinovirus (RV) infected bronchial epithelial cells (133.44)
Authors: Wark Peter AB , See Hayley V , Simpson Jodie L , Forbes Rebecca L , Hansbro Phil
Published: January 2023

Rhinovirus (RV) infection is an important trigger of acute asthma and COPD.

Study objectives; were to characterise the response of PBMCs and CD8 lymphocytes when exposed to RV infected bronchial epithelial cells.

Methods; Human Calu3 cells were used as a model for bronchial epithelial cells and infected with RV-43 and RV-1B. PBMCs were obtained from healthy individuals and separated by Ficoll-Paque centrifugation. CD8 lymphocytes were identified as CD3+, CD8+ by flow cytometry. PBMCs were exposed to infected Calu3 cells, cells exposed to UV inactive RV, PHA and TLR3 agonist PolyI:C. Response was measured by ELISA for release of interferon(IFN)-γ and TNF-α. CD8 activation with expression of perforin or proliferation measured by carboxyfluorescein succinimidyl ester assay.

Results: Exposure of PBMCs to Calu3 cells infected with both RV led to significant release of IFN-γ and TNF-α, smaller release was seen in response to UV inactive RV. Exposure to PolyI:C also led to a similar release. CD8+ cells were found to be the primary source of IFN-γ release. CD8+ cells did not show an increase in perforin expression or evidence of proliferation.

Conclusions; exposure of PBMCs and CD8+ cells to an RV infected bronchial epithelium alone results in an innate immune response mediated by TLR3 without evidence of a CD8 adaptive immune response.
TLR-7 Mediated Responses to Respiratory Syncytial Virus (RSV) (133.45)
Authors: Lindell Dennis M , Morris Susan , Mukherjee Sumanta , Lukacs Nicholas W
Published: January 2023

Respiratory syncytial virus (RSV) infection results in lung disease characterized by mucus hypersecretion and inflammation. RSV has also been implicated in both the initial development of asthma, as well as the exacerbation of existing asthma. Previous studies in our laboratory have demonstrated roles for TLR3 and the adapter molecule MyD88 in the host response to RSV. In these studies, we investigated the role of TLR 7 in the recognition of RSV and RSV-induced pulmonary disease. TLR7 -/- and WT C57 Bl/6 mice were infected intratracheally with 10^5 PFU RSV Line 19. Compared to control mice, TLR 7-/- mice had significantly increased mucus hypersecretion, as assessed via PAS staining of lung sections and the expression of Muc5ac and Gob5. Mucus hyperresponsiveness was associated with increased IL-17 and IL-13 (as well as other cytokines). Cell type specific studies revealed altered responses to RSV in TLR7 -/- B cells, plasmacytoid dendritic cells (pDC), and surprisingly, classical myeloid dendritic cells (mDC). Studies in bone marrow-derived dendritic cells revealed TLR-7 dependent effects on IL-12 signaling. In response to the TLR7/8 specific agonist 3M-011, BMDC dramatically up-regulated IL-12 p35. In response to RSV, TLR7 -/- BMDC were significantly impaired in the induction of IL-12 p35 in response to RSV, but exhibited significantly higher IL-23 p19. Thus, TLR-7 regulates IL-12/IL-23 responsiveness to RSV.

Supported by P01HL031963 (NWL), R01AI073876 (NWL)
Evidence of Murine Alternatively Activated Lung Macrophages are Associated with Failure to Clear Pulmonary Cryptococcus neoformans (133.54)
Authors: Quintero Eric A , Gott Katherine A , Wilder Julie A
Published: January 2023

We have previously shown that murine pulmonary immunity to the fungal pathogen Cryptococcus neoformans (Cne) is genetically regulated. C.B-17 mice efficiently clear the organism from the lung following intranasal inoculation whereas C57BL/6 mice do not. The ability to clear Cne from the lungs of C.B-17 mice is dependent upon the development of a Type 1 immune response. In contrast, infected C57BL/6 mice mount a Type 2 immune response. As alternatively activated macrophages (AAMs) have been associated with the development of a Type 2 immune response, we reasoned that Cne-infected C57BL/6 mice may have more AAMs in their lungs as compared to infected C.B-17 mice. We tested our hypothesis by analyzing the expression of four transcripts generally over-expressed in AAMs (YM1, FIZZ1, ARG1, and ARG2) in whole lung homogenate samples via PCR. Our data show increased expression of these markers over time post-Cne infection in both strains (increasing from 6 hours - 14 days) with greater expression in C57BL/6 mice at all time points. Lungs from naïve C57BL/6 mice also showed higher expression of ARG1, FIZZ1 and YM1 compared to naïve C.B-17 lungs. These data support our hypothesis that there are more AAMs in the lungs of Cne-infected C57BL/6 mice as compared to similarly infected C.B-17 mice. This research was supported by Lovelace Respiratory Research Institute internal funds.
Bacillus anthracis lethal toxin abrogates TCR-induced cytokine production by CD1d-restricted Natural killer like - T cells (134.15)
Authors: Joshi Sunil K , Lang Gillian , Devera T. Scott , Aye Lindsay M , Shah Hemangi B , Ballard Jimmy , Lang Mark
Published: January 2023

Exogenous CD1d-binding glycolipid (α-Galactosylceramide, α-GC) stimulates TCR signaling and activation and proliferation of type-1 natural killer like - T (NKT) cells. These activated NKT cells play a central role in regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the direct in vivo and in vitro effect of Bacillus anthracis lethal toxin (LT) on NKT cells. LT is a complex protein formed by pore-forming protective antigen (PA) upon binary combination with lethal factor (LF) subunit. A sub-lethal dose of LT administered in vivo in C57BL/6 mice altered expression of the activation receptor NKG2D by NKT cells but not by NK cells. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound PA more than other immune cell types. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. We propose that Bacillus anthracis-derived LT contributes to immune evasion by altering NKT function.

This work was supported by Defense Threat Reduction Agency (DTRA) grant CBDIF-07-PRET-01 to M.L.L and by a pilot award to M.L.L. as part of NIH grant 5 U19 AI062629-05.
Post-translational regulation of the NKG2D ligand Mult1 in response to cell stess (134.2)
Authors: Nice Timothy J , Coscoy Laurent , Raulet David H
Published: January 2023

NKG2D is a major stimulatory receptor expressed by natural killer cells and some T cells. The receptor recognizes MHC class I-like cell surface ligands that are poorly expressed by normal tissues but often induced in transformed and infected cells. Induction of NKG2D ligands during transformation or infection leads to activation of NKG2D-expressing immune cells and elimination of the diseased target cells. The existence of several NKG2D ligands in each individual, some with strikingly divergent protein sequences, raises the possibility that different ligands are regulated by distinct disease-associated stresses. The transcripts for some ligands, including murine UL16-binding protein-like transcript 1 (Mult1), are abundant in certain normal tissues where cell surface expression is absent suggesting the existence of translational or post-translational regulation. We report that under normal conditions, Mult1 protein undergoes ubiquitination dependent on lysines in its cytoplasmic tail and is degraded by the lysosome. Mult1 degradation and ubiquitination is reduced in response to stress imparted by heat shock or ultraviolet (UV) irradiation, but not by other forms of genotoxicity, providing a novel mechanism for stress-mediated cellular control of NKG2D ligand expression.
TLR3 Signaling in iNKT cell Promotes Apoptosis of γδ T cells (134.27)
Authors: Ajuebor Maureen N , Jin Yijun , Chen Qingling , Gardner Tommy
Published: January 2023

Polyinosinic-polycytidylic acid (poly I:C, a specific ligand for TLR3) activate immune cells (such as DC and NK cell) in a TLR3-dependent manner to induce cytokine production. TLR3 expression by iNKT cells has not been determined. The main objective of this study was to determine TLR3 expression by iNKT cell and their functional responsiveness after stimulation with poly I:C. We hypothesize that TLR3 signaling in iNKT cells suppress γδT cell functions by induction of apoptosis. Our hypothesis is based on the following observations: First, poly I:C administration into C57BL6 mice was associated with extracellular TLR3 and intracellular cytokine (IFN-γ and TNF-α) expression by hepatic iNKT cells. Second, activated hepatic iNKT cells promote apoptosis of γδT cells via induction of IFN-γ and TNF-α since pretreatment of C57BL6 mice with anti-TNF-α or anti-IFN-γ Abs prior to poly I:C administration caused a significant decrease in the frequency of active caspase 3 expression by hepatic γδT cells. Finally, the frequency of γδT cells accumulation in the liver was significantly increased by iNKT cell deficiency (vs. WT mice) after poly I:C administration. Moreover, hepatic γδT cells derived from iNKT cell KO mice were resistant to apoptotic death (and thus associated with increased survival) relative to hepatic γδT cells derived from WT mice post-poly I:C administration. It is noteworthy that serum levels of TNF-α and IFN-γ were also ablated by iNKT cell deficiency. In summary, our results support the notion that (i) hepatic iNKT cells directly respond to TLR3 signaling to activate γδT cells; (ii) the activation depends on cell-cell contact whereby iNKT cell-derived cytokines induce the programmed cell death of γδT cells; an effect that may attenuate potential effector functions of γδT cells.
Autocrine signaling produced by M2bMφ is required for the prolongation of properties (134.29)
Authors: Asai Akira , Shigematsu Kenji , Kobayashi Makiko , Herndon David N , Suzuki Fujio
Published: January 2023

Three different subtypes of M2Mφ (M2aMφ, M2bMφ and M2cMφ) have recently been demonstrated in mice with severe burn injuries. M2aMφ and M2cMφ appeared 1 to 4 days after burn injury, while M2bMφ appeared in mice 1 to 3 weeks after burn injury. In this study, how M2bMφ with suppressor cell activities are prolonged is examined. M2bMφ were peritoneal Mφ isolated from burned BALB/c mice (25% TBSA, 3rd degree flame burn) using biotin-conjugated anti-mouse F4/80 mAb and streptavidin magnetic beads. M2bMφ (1 x 106 cells/ml) isolated from mice 16 days after burn injury were cultured under frequent medium changes (every 12 hrs for 3 days). As a control, the same M2bMφ preparation was cultured for 3 days without any medium changes. Mφ harvested were assayed for LIGHT mRNA by RT-PCR. Also, obtained Mφ were cultured an additional 120 hrs and culture fluids harvested were assayed for CCL1 using ELISA. LIGHT mRNA and CCL1 were typical biomarkers for M2bMφ. In the results, M2bMφ cultured for 3 days without any medium changes expressed LIGHT mRNA. However, this mRNA was not expressed by M2bMφ cultured with frequent medium changes. CCL1 was detected in the culture fluids of M2bMφ that had been previously cultured without any medium changes. When M2bMφ were cultured for 3 days under the frequent medium changes, the ability of these cells to produce CCL1 was reduced by 70%. These results indicate that M2bMφ cultured under the frequent medium changes lose their M2bMφ properties. Some soluble factors in the culture fluids of M2bMφ were shown to be involved in the persistence of M2bMφ properties. This study was supported by SHC NA #8620.
Heterogeneity of COX-2 macrophages in acute and chronic mycobacterial inflammation (134.30)
Authors: Shibata Yoshimi , Kogiso Mari , Pantuso Traci , Shinohara Tsutomu , Dorey C. Kathleen , Henriksen Ruth Ann
Published: January 2023

Persistent up-regulation of cyclooxygenase-2 (COX-2) and over-production of PGE2 by activated macrophages (MØ) compromise host immunity in cancer immunotherapy, autoimmune diseases, and chronic mycobacterial inflammation such as TB. Previous studies showed that a population of COX-2+ MØ is induced in the spleen of mice given i.p. HK-Mycobacterium bovis BCG. These MØ are derived from radiosensitive immature bone marrow cells that migrate and localize in the spleen. Mature spleen MØ express active COX-2 (subcellularly associated with the nuclear envelope (NE)), and release increased PGE2 from day 5 to day 21 after BCG administration. To determine if local sites of inflammation caused by BCG accumulate similar COX-2+ MØ, C57Bl/6 mice were given i.p. or i.n. BCG (1 and 0.5 mg doses, respectively). We found that 1 - 2 days after i.p. administration, peritoneal and splenic MØ transiently exhibited COX-2 which was dissociated from the NE, inactive, and released no PGE2. However, unlike splenic MØ, neither active nor inactive COX-2 was found in peritoneal MØ over days 3 - 28. After i.n. administration, alveolar MØ persistently expressed inactive COX-2 over days 1 - 28. No alveolar MØ with active COX-2+ were detected. Normal splenic, peritoneal, and alveolar MØ treated in vitro with BCG expressed active COX-2 and increased PGE2 release within 24 h, indicating that in vitro MØ activation does not always mimic in vivo responses. Our results indicate that in vivo BCG preferentially induces active COX-2 in the splenic MØ. In addition, without suppression by PGE2, MØ with inactive COX-2 may be "hyper-active" and regulate tissue-dependent acute and chronic inflammation. (Supported by NIH HL71711, DOD DAMD 17-03-1-0004, BCCRP 06BS-04-9615)
Serum Amyloid P inhibits Renal Fibrosis through an Fc-gamma receptor dependent mechanism. (134.33)
Authors: CASTANO ANA P , Lupher Jr Mark , Duffield Jeremy S
Published: January 2023

Serum amyloid P (SAP) inhibits fibrocyte outgrowth from PBMCs and may therefore play a role in fibrosis. We studied the role of SAP in regulating fibrosis in the kidney where there are very few fibrocytes. In the UUO model of kidney fibrosis, IP or IV SAP (20µg/g) significantly inhibits fibrosis. (11.3% at 14d vs. 5.4%). Similar findings were seen in other models of kidney fibrosis. In transgenic Col1-GFP mice, SAP reduced myofibroblast GFP by 50%, but myofibroblast numbers were not affected, indicating SAP switches off collagen production. SAP does not bind to fibroblasts or reduce collagen production by fibroblasts in vitro. SAP binds to monocytes, Møs and apoptotic cells. Møs are fibrogenic through paracrine signaling to fibroblasts. SAP binds FcγRI, II, III, IV, in mice and inhibits Monocyte/Mø activation, assessed by inflammatory gene transcript levels, and cytokine production in vitro. Primed Møs are susceptible to soluble IgG-mediated activation suggesting SAP inhibits kidney Mø activation by an Fcγ-dependent mechanism. Purified UUO kidney Møs were also less activated and produced more IL10 in mice treated with SAP. To test the role of FcγRs in kidney Mø activation we induced UUO fibrosis in FcRγ-chain KO mice. Compared with controls, fibrosis was attenuated, confirming a role for FcγR-mediated activation of kidney Møs in the development of fibrosis. Our studies point to SAP as a potential therapeutic in the management of progressive fibrosis.
Defective Dendritic Cell Phagocytic Function in Aging (134.36)
Authors: Julius Annabelle , Abernathy Lisa , Yung Raymond
Published: January 2023

Dendritic cells (DCs) play an important role in many immune-mediated processes in aging. One of their major functions is the phagocytic clearance of pathogens and tumor cells. Currently, very little is known about the role of aging in determining DC phagocytic function. Using murine CD11c+ bone marrow-derived DCs from young (3-6 months) and old (21-23 months) C57BL/6 mice, we compared the phagocytic function of DCs. We found that old DCs have decrease capacity to internalize exogenous FITC-dextran compared with young DCs. Similarly, old DCs displayed a decrease in the global uptake of E. coli bacteria per cells (398 ± 32.6 fluorescence units) when compared to young DCs (598.7 ± 13.7). This impaired phagocytic activity in old DCs was further confirmed by quantifying the number of bacteria ingested per cell by confocal microscopy. Intriguingly, aging does not affect the ability of DCs to ingest zymosan or Gram positive bacteria such as S. Aureus, suggesting a decrease in the expression and/or function of selective receptors associated with gram negative bacteria phagocytosis in aging. Cell surface expression analysis of several receptors including CD206, CD205, TLR2, TLR4 and TLR5 were similar in both aged group. However, aging impacts the expression of several mediators of TLR4 Myd88 independent but not dependent signaling, including Ticam2 and IRF3, which may contribute to the observed impaired gram negative DC-mediated phagocytosis in aging. A better understanding of the effect of age on DC phagocytosis may improve our understanding on why selected infectious diseases are more prevalent in the elderly and provide us with new treatment options.
Thapsigargin and flavin adenine dinucleotide ex vivo treatment improves leukocyte microbiocidal activity through rescuing trafficking-defective gp91-phox in chronic granulomatous disease (134.37)
Authors: Huang Ya-Fang , Liu Si-Yen , Yang Pei-Wen , Yen Chia-Liang , Shieh Chi-Chang
Published: January 2023

Mutations in leukocyte NADPH oxidase genes lead to defective respiratory burst in white blood cells and cause chronic granulomatous diseases (CGD) in affected patients. The most common form of CGD is caused by mutations in the membrane-bound component of gp91-phox, which is encoded by the CYBB gene located on the X chromosome. We previously reported a patient with CYBB mutation (H338 to Y) which prevents the intracellular trafficking and expression of gp91-phox on the leukocyte surface. By treating the leukocytes with thapsigargin and flavin adenine dinucleotide (FAD), we rescued the capacity of the leukocytes to produce reactive oxygen species (ROS). We visualized the enhanced cell surface expression of the mutant protein after thapsigargin and FAD treatment with GFP-tagged gp91-phox expression with the CGD mutation. Protein and cellular analyses showed that treated cells expressed more endo-H resistant gp91-phox protein and are more effective in killing bacteria. Thapsigargin and FAD-treated CGD leukocytes had enhanced activity in protecting mice from S. aureus-induced peritoneal abscess formation in a mouse model of CGD. These results indicate that thapsigargin and FAD ex vivo treatment is effective in rescuing the ROS-producing activity of leukocytes in selected CGD patients.
Modeling the Maturation Process of Dendritic Cells (134.40)
Authors: Gonzalez Joanna , Moran Thomas M , Sealfon Stuart C
Published: January 2023

Dendritic cells are antigen-presenting cells responsible for the initiation of immunity. Immature DCs act as sentinels that, upon recognizing a pathogen, alter their gene expression pattern; the mature DCs then secrete signals to stimulate the appropriate immune response. One of these signals is the cytokine IL-12, composed of IL-12p35 and IL-12p40; the interaction of both subunits is required for the production of bioactive IL-12. A related cytokine, IL-23, is formed by the interaction of IL-12p40 with IL-23p19. The relative proportions of IL-23 and IL-12 depend on the pathogen recognized by the DC. Consequently, a model is being built in order to attain a more accurate understanding of IL-12 synthesis. The first part of the model encompasses the transcriptional activation of IL-12 genes. Previous research has identified some of the transcription factors involved and it has been found that the transcriptional regulation of IL-12 and IL-23 involves both common and distinct signaling pathways. However, further experiments are required to obtain a more complete signaling network from virus infection to the activation of these transcription factors. The second part of the IL-12 model involves the synthesis of bioactive IL-12 and mass-action kinetic equations have been developed to approximate the transcription and translation of the two subunits, their dimerization and secretion. Experimental data are currently being obtained for kinetic parameter estimation, as well as for testing the model; this proposed methodology can then be extended to other DC maturation signals in order to acquire a more complete understanding of the initiation of immunity.
Ibuprofen, the popular over-the counter non-steroidal anti-inflammatory drug, blunts human antibody synthesis in vitro (134.41)
Authors: Bancos Simona , Phipps Richard P.
Published: January 2023

Background: Non-steroidal anti-inflammatory drugs (NSAIDs) are effective in reducing fever, relieving pain and inhibiting inflammation. NSAIDs function through inhibition of cyclooxygenases 1 and 2 (Cox-1 and Cox-2). Cox-1 is ubiquitously expressed, while Cox-2 expression is increased during inflammation and in malignant cells. We recently showed that Cox-2 is up-regulated in normal human B lymphocytes after activation and is required for optimal antibody synthesis. We hypothesized that ibuprofen (dual Cox-1/Cox-2 inhibitor) blunts antibody synthesis in human peripheral blood mononuclear cells (PBMC) and B lymphocytes.

Methods: IgM and IgG production in anti-IgM + CpG 2395 stimulated human PBMC and B cells was detected by enzyme-linked immunosorbent assay (ELISA). B cell proliferation and viability in the presence of ibuprofen was calculated using mitochondrial dehydrogenase activity (MTT) and 3H-Thymidine incorporation assays.

Results: Pharmacological concentrations of ibuprofen significantly reduced IgM and IgG synthesis in human PBMC and B lymphocytes in a dose-dependent manner. Ibuprofen had only a modest effect on B cell viability, but significantly inhibited B cell proliferation.

Conclusion: Ibuprofen reduces optimal antibody synthesis in part, by decreasing B cell proliferation.

Research supported by DE011390, AI071064, ES01247 grants.
Immunomodulatory activity of synthetic innate defence regulators (IDRs) (134.45)
Authors: Easton Donna M , Ma Shuhua , Mookherjee Neeloffer , Hamill Pamela , Lynn David , Gardy Jennifer , Mullaly Sarah , Pilat Sandra , Waldbrook Matthew , Thorson Lisa , Falsafi Reza , Chikatamarla Avinash , Team InnateDB , Schofield Louis , Finlay Brett , Brinkman Fiona , Hancock Robert EW
Published: January 2023

Cationic host defence (antimicrobial) peptides, e.g. cathelicidin LL-37, have a variety of immunomodulatory activities that favour the safe resolution of infections. We have studied novel synthetic cationic innate defence regulator peptides that are not directly antimicrobial but are anti-infective in vivo, due to modulation of innate immunity. A range of peptides derived from the small bovine peptide bactenecin were screened for immunomodulatory activities in vitro; e.g. promotion of chemokine production and suppression of pro-inflammatory cytokines. Since innate immunity is complex, involving >1,500 gene products, a systems biology approach was utilized to characterize peptide modulation of innate immunity, including analysis of receptors, signalling pathways, transcription factors and downstream genes. To permit visualization and bioinformatic analysis of complex events, an innate immunity database (www.innatedb.ca), a network visualization tool (Cerebral) and downstream analysis tools (e.g. pathway overrepresentation analysis) were developed and provided insight into how this selective modulation occurs. In vivo data indicate that these activities provide protection in animal model infections of Gram positive and Gram negative bacterial infections as well as severe malaria. Thus IDRs have great potential for use as novel anti-infective agents.

Supported by Genome BC, FNIH and CIHR.