bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunogenicity and specificity of Norovirus Consensus GII.4 virus-like particles in monovalent and bivalent vaccine formulations (113.27)
Authors: Parra Gabriel , Bok Karin , Taylor Ross , Haynes Joel , Sosnovtsev Stanislav , Richardson Charles , Green Kim
Published: January 2023

Noroviruses, a major cause of acute gastroenteritis worldwide, present antigenic diversity that must be considered for the development of an effective vaccine. In this study, we explored approaches to increase the broad reactivity of virus-like particle (VLP) norovirus vaccine candidates. The immunogenicity of a GII.4 “Consensus” VLP that was engineered from sequences of three distinct naturally-occurring GII.4 strains was examined for its ability to induce cross-reactive immune responses against different noroviruses. Rabbits immunized with GII.4 Consensus VLPs developed high serum antibody titers against VLPs derived from a number of distinct wild-type GII.4 viruses Because the sera exhibited low cross-reactivity with antigenically-distinct GI norovirus strains, we investigated the serum antibody response to a bivalent vaccine formulation containing GI.1 and GII.4 Consensus VLPs that was administered to animals under varying conditions. In these studies, the highest homologous and heterologous antibody titers to the bivalent vaccine were elicited following immunization of animals by the IM route using Alhydrogel as adjuvant. Our data indicate that the use of both genetically-engineered norovirus VLPs that incorporate relevant epitopes from multiple strains and multivalent vaccine formulations increase the breadth of the immune response to diverse variants within a genotype and, thus, prove helpful in the rational design of VLP-based vaccines against human noroviruses.
Non-inflammatory and additive-free adjuvant for cutaneous vaccination (113.29)
Authors: Wu Mei , Shah Dilip , Chen Xinyuan , Anderson Richard
Published: January 2023

Skin has been considered an attractive site for vaccine delivery for more than 3000 years, but it is still not commonly used today, because of technical difficulties and a lack of inflammation-free adjuvant. Considerable progress made in generating convenient intradermal (i.d.) injection devices in the past decade raises an urgent need of developing potent vaccine adjuvants that introduce little skin inflammation. Many current vaccine adjuvants cause severe skin inflammation that jeopardizes the integrity of the skin and thus cannot be accepted for cutaneous vaccination. We develop a laser-based vaccine adjuvant (LVA) capable of boosting immune responses without incurring inflammation. LVA was induced by brief (2 min) illumination of a small area of the skin with a safe laser prior to i.d. administration of vaccines at the site of laser illumination. The pre-illumination augmented the hemagglutination inhibition (HAI) titers against seasonal or 2009-pandemic influenza vaccine by 10~20-folds when compared to i.m. immunization. And, while i.d. immunization blocked viral production in the lung by 2-fold over i.m. vaccination, i.d. immunization after laser illumination increased the blockade to 186-fold. In comparison with all current vaccine adjuvants that are either chemical compounds or biological agents, LVA is a risk- and additive-free adjuvant and has distinct advantages over traditional vaccine adjuvants for cutaneous vaccination.
Vaccine Induced Protection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection in swine (113.3)
Authors: Hemnani Khushboo , Trujillo Jessie , Opriessnig Tanja , Messel Robert , Tobin Gregory , Nara Peter
Published: January 2023

PRRSV is an economically significant Arterivirus of swine health industry. High genetic diversity (up to 20% strain variability), high mutation rate and various immune evasion strategies pose major challenges in the development of an efficacious broadly protective vaccine. One evasion strategy is the presence of non-conserved immunodominant decoy epitopes that divert the immune response towards non-protective, type-specific ones. We hypothesize that masking decoy epitopes with PRRSV specific antibodies, Antibody Immune Masking (AIM), can redirect immune response towards broadly protective neutralizing antibodies. Presented here is the development of an efficacious AIM PRRSV vaccine model capable of inducing sterilizing immunity in some vaccinated pigs and reduction of viremia as well as PRRSV associated pathology in infected pigs. Pigs were vaccinated with AIM or autologous vaccine and later challenged with a homologous/heterologous virus. Results from a trial with 10 pigs per group demonstrated that AIM vaccination was effective in prevention of heterologous infection in 33% of pigs, while others had significant reduction in peak viremia as compared to the autogenous vaccination control pigs (p=0.0034) and unvaccinated controls (p=0.0001); as well as reduction in pulmonary viral loads compared to unvaccinated controls (p=0.0126). Current research is focused on determination and characterization of correlates of immune protection and confirmation of immune refocusing.
Seasonal IM flu-vaccines boost antibody titers to epitopes common to influenza virus strains (113.35)
Authors: Kasten-Jolly Jane , Lawrence David
Published: January 2023

Human serum IgG anti-influenza virus antibodies (Abs) to components of the inactivated, tri-valent 2006/2007, 2007/2008, 2008/2009, and 2010/2011 vaccines were measured by surface plasmon resonance (SPR; BIA3000). Pre- and post (2 wk) 2007/2008 vaccination (N=20 healthy volunteers) serum IgGs were assayed for binding to the 2006/2007, 2007/2008, and 2008/2009 vaccine preparations. Overall, the IgG binding was greatest to the 2006/2007 vaccine components, but binding was also observed against the 2008/2009 vaccine which did not include strains within the 2006/2007 or the 2007/2008 vaccines. Several control individuals who had never received a flu-vaccine showed no binding to any of the vaccines studied. Pre- and post (2 wk) 2010/2011(H1N1, pandemic strain) vaccination Abs were assayed for binding against the 2006/2007, 2007/2008, and the 2010/2011 vaccines. Vaccination with the 2010/2011 vaccine showed a >10 fold boost in IgG binding to both the 2006/2007 and 2010/2011 components and somewhat lower to 2007/2008, compared to the 2-3 fold boosts to the 2006/2007 components observed with the previous vaccinations. The results emphasize that Abs raised against one vaccine preparation can bind strains present in other vaccine preparations. The 2006/2007 vaccine preparation seems to have one or more of these highly antigenic epitopes. Future studies will involve delineation of these sites and investigation into their ability to protect against influenza viral infection.
Long term persistence of IgE anti-hepatitis A virus antibodies in adult serum post vaccination. (113.9)
Authors: Bluth Martin , Kokh Dina , Zhou Weili , Rirash Fadumo , Smith-Norowitz Tamar
Published: January 2023

Rationale: The role of IgE in immunity against Hepatitis A has not been studied. Methods. Serum was obtained from an adult who was vaccinated with Hepatitis A virus (HAV) (Havrix®) in 2005 and again in 2009 and compared with pre vaccination status and natural HAV infection. Serum was assessed for the presence of IgE anti-HAV antibody (modified Immunoblot/ELISA, spectrophotometry) over seven years post vaccination and compared with IgG and IgM virus specific antibodies.. Results. Serum obtained from HAV adult contained IgE anti-virus antibodies which increased over 1.5 years post initial vaccination. In contrast, serum levels of IgE anti-HAV antibodies increased 6 fold two years after 2nd vaccine booster compared with pre-booster levels (1.2OD450 vs 0.21OD450, p<0.05). The rise of IgE anti-HAV levels increased proportionally with time after booster. Interestingly, serum IgE HAV antibody levels seven years post vaccination were comparable to levels of IgE anti-hepatitis antibodies in natural HAV infection (1.2OD450 vs 1.23OD450). Similar patterns were not observed with IgG and IgM anti virus antibodies with respect to consistency and rate of rise. Conclusions. This is the first description of the presence of IgE anti-HAV antibodies, and the first demonstration of its long term persistence serum seven years post vaccination. Understanding the role of IgE anti-virus antibodies may provide insight into vaccine physiology with respect to protection against viral infection.
The CLRX.1/NOD24 pseudogene codes a functional NF-κB regulator, Pyrin-only protein 3 (POP3) (114.10)
Authors: Harton Jonathan , Atianand Maninjay , Porter Kristen
Published: January 2023

Inflammasomes play a crucial role in mediating innate and adaptive immunity against a variety of microbes and danger signals. However, regulatory mechanisms controlling inflammasome effector functions, specifically generation of the bioactive IL-1β in macrophages are not well studied. Pyrin-only proteins (POPs) modulate NF-κB signaling and inflammasome activation, thus restricting the magnitude of innate immune and inflammatory responses in humans. POP1 interferes with IKKα/β activation thus limiting NF-κB activity with no known effect on ASC-dependent inflammasomes. POP2, however, limits the transcriptional activity of NF-κB p65/RelA while also down-modulating the assembly of various ASC-dependent inflammasomes. Here we examine a third human POP, POP3, the product of an NLRP pseudogene with ubiquitous and inducible expression. Like POP1 and POP2, POP3 impairs NF-κB-mediated production of pro-inflammatory cytokines, but fails to inhibit ASC-dependent inflammasomes likely due to the absence of specific acidic residues within its first α helix. Despite high homology to POP2, POP3 is translocated to the nucleus in parallel with NF-kB and inhibits the transcriptional activity of nuclear NF-kB. Collectively, POP3 is a resurrected processed-pseudogene that in addition to other POPs likely represents a further level of control for NF-κB-dependent inflammatory responses
NLRP3 inflammasome dependent loss of mitochondrial and cell membrane integrity in bone marrow derived macrophages (114.3)
Authors: Heid Michelle , Maniar Salony , Watkins Simon , Salter Russell
Published: January 2023

The NLRP3 inflammasome is a multiprotein complex that includes the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which binds with caspase-1 via CARD-CARD interactions and is involved with caspase-1 activation. Pore forming toxins, such as tetanolysin O (TLO), as well as the potassium ionophore nigericin, stimulate the NLRP3 inflammasome. We have observed that NLRP3 knockout macrophages are more resistant to the loss of membrane integrity compared to wild type macrophages exposed to nigericin or TLO, as assessed by a dye uptake assay. Furthermore, this NLRP3 dependent loss of membrane integrity corresponds to the loss of mitochondrial membrane integrity as demonstrated via live cell microscopy. The loss of mitochondrial membrane integrity, but not mitochondrial depolarization was found to be dependent on the presence of NLRP3. Furthermore, the loss of mitochondrial integrity and the loss of cell membrane integrity suggest a potential mechanistic pathway for the effects of NLRP3 inflammasome activation in BMDM. These results further our understanding of how NLRP3 functions in macrophages exposed to stressful stimuli such as bacterial toxins.
Intrathymic programming of effector fates in three molecularly distinct γδ T cell subtypes (115.16)
Authors: Narayan Kavitha , Sylvia Katelyn , Malhotra Nidhi , Yin Catherine , Martens Gregory , Vallerskog Therese , Kornfeld Hardy , Xiong Na , Cohen Nadia , Brenner Michael , Berg Leslie , Kang Joonsoo , Immunological Genome Project Consortium The
Published: January 2023

Two distinct lineages of T cells, distinguished by expression of either an αβ or γδ T cell receptor (TCR), arise from a common progenitor in the thymus. The type of pathogen and the cytokine milieu directs effector differentiation of αβ T cells in the periphery through the induction of specific transcriptional networks. γδ T cell development is distinct from that of αβ T cells in its ordered rearrangement of TCR genes and the pairing of Vγ and Vδ chains to generate γδ T cell subsets that home to specific tissues. While γδ T cells have primarily been studied as one homogenous population, they can be functionally classified into effector subsets based on the production of signature cytokines, analogous to αβ T helper subsets. Unlike αβ T cells, γδ T cell effector function correlates with genomically encoded TCR chains, but the mechanism of γδ effector specification is unclear. In conjunction with the Immunological Genome Project Consortium, we performed a high resolution transcriptome analysis of all emergent γδ thymocyte subsets segregated based on TCRγ/δ chain usage and maturation state in the thymus. Our data indicate the existence of three separate subtypes of γδ T cells in the thymus. The profiles of the emergent immature γδ T cell subsets are already embedded with unique gene programs directing subset-specific effector function, indicating that γδ T cell effector function is molecularly programmed in the thymus prior to, or concurrent with, TCR expression.
Metabolic profiling of T-cell differentiation and tolerance (115.5)
Authors: Kissick Haydn , Finke Jorge , Dunn Laura , Asara John , Sanda Martin , Arredouani M.Simo
Published: January 2023

Metabolic pathways that drive T cell differentiation, activation and tolerance remain poorly understood. Here we used mass spectrometry to partially describe the metabolic profile of CD4 and CD8 T cells that underwent in vitro activation and differentiation in the presence or absence of prostate cancer conditioned media. T cell subsets were purified from mouse spleen or human PBMC, activated in vitro in the presence of various combinations of cytokines or prostate cancer cell line conditioned media. Metabolites were extracted using methanol, injected and analyzed using a 5500 QTRAP triple quadruple mass spectrometer coupled to a Prominence UFLC HPLC system via selected reaction monitoring (SRM) of a total of 255 endogenous water soluble metabolites for steady-state analyses of samples. Our data show that TCR/CD28-mediated activation imposes a distinct metabolic profile on mouse T cells. This profile is distinct for each cell lineage when activated cells are differentiated into either Th1, Th2, Th9, Th17 or regulatory T cell phenotypes. Likewise, metabolic profiles of activated CD4 and CD8 T cells from both mouse and human are significantly affected by the presence of prostate cancer cell line conditioned media, with major changes in the urea cycle, ammonia recycling, and protein and nucleotide biosynthesis . Together, our findings provide a basis for further development of drugs that could be used to manipulate T cells for a better outcome of prostate cancer vaccines.
microRNA miR-155 regulates NKT cell development and function by targeting ITK (115.6)
Authors: Zhou Li , Li Kai , Zheng Quanhui , Seo Kook-Heon , He Hong-Zhi , Mi Qing-Sheng
Published: January 2023

Invariant Natural Killer T (iNKT) cells are potent regulators of diverse immune responses. Our previous study indicated that lack of miRNAs following the deletion of Dicer, a miRNAs-processing enzyme, affect iNKT cell development and function. However, the roles of specific miRNA in iNKT cell development and function has not been reported. To identify which miRNAs are involved in NKT cell development, we first performed a genome-wide miRNA expression profiling during NKT cell development and found that miR-155 was highly expressed in early iNKT cell development, followed by a sharp decrease in late developmental stage in thymus. To test the role of miR-155 in iNKT cells, two miR155 mutant mouse models had been used. With a conventional miR-155 deficient mouse model, we did not find remarkable defects of iNKT cell development and function. However, overexpression of miR-155 in bone marrow or thymus results in iNKT cell developmental defect in the thymus without dramatic effects on other immune cells. Furthermore, miR-155 overexpressed iNKT cells display profound defects in their cytokine production. Bone marrow from miR-155 overexpressed mice poorly reconstituted iNKT cells, suggesting a cell intrinsic effect. Finally, we have identified Itk (Inducible T cell Kinase) as a direct target of miR-155 in the regulation of iNKT cell development and function. Together, our data suggest that dynamic regulation of miR-155 expression is required for iNKT cell development and function.
Requirement of miR155 for NK cell and CD8+ T cell responses against viral infection (115.7)
Authors: Sun Joseph , Zawislak Carolyn , Loeb Gabriel , Beaulieu Aimee , Bezman Natalie , Lanier Lewis
Published: January 2023

Natural killer (NK) cells and CD8+ T cells function similarly in the recognition and destruction of host cells infected with pathogens. Although microRNAs (miRs) are critical for NK and T cell development and function, the role of specific miRs remain to be investigated. We discovered that miR155 is induced in activated NK cells during mouse cytomegalovirus (MCMV) infection. Using mice containing a targeted deletion of miR155, we determined that miR155 is indispensible for MCMV-specific NK cell expansion and generation of long-lived NK cells. Although miR155-deficient NK cells do not have any defects in basal NK cell homeostasis or function, they exhibit severely impaired effector and memory cell numbers in both lymphoid and non-lymphoid tissues following viral infection. Furthermore, miR155-deficient CD8+ T cells are similarly compromised in their ability to undergo clonal expansion and survive following MCMV infection, together resulting in substantially diminished memory cell numbers. Thus, miR155 is essential for the generation of protective effector and memory NK and CD8+ T cells during viral infection.
A small peptide binds to pathogenic effector cells to prevent/reverse hyperglycemia in T1D (116.1)
Authors: Wagner David , Olmstead Michael
Published: January 2023

CD40 plays a prominent role in autoimmunity. We described its expression on CD4 cells termed CD4CD40 cells. Diabetogenic T cell clones are CD40+ while non-diabetogenic clones are CD40-. CD4CD40 cells expand in number in NOD mice developing progressive insulitis and hyperglycemia and are necessary for adoptive transfer of T1D to NOD.scid recipients. CD4CD40 cells are found in human subjects, significantly (p< 10-7) increased in T1D, including new onset and long term (> 40 yrs) T1D subjects. CD4CD40 cells are expanded regardless of HLA haplotype but occur at normal levels in non-diabetics even those carrying predictive HLAs. CD4CD40 cells are significantly increased in diagnosed pre-diabetic subjects suggesting a biomarker for T1D diagnosis. CD4CD40 cells produce predominantly Th1 cytokines but also produce IL-17 suggesting an intermediary between Th1 and Th17 cells. Blocking CD40 - CD154 with anti-CD154 antibody prevents diabetes and contains CD4CD40 cell expansions. A 15 amino acid peptide derived from the CD154 protein sequence binds directly to CD4CD40 cells and prevents hyperglycemia onset. Furthermore the peptide reverses hyperglycemia in new onset T1D mice. This peptide constricts CD4CD40 cell numbers similarly to anti-CD154 treatment but does not disrupt immune homeostasis. Peptide treated mice have less islet infiltrates and greater insulin production than controls. Peptide treated mice also have increases in FoxP3+ cells, suggesting a tolerogenic mechanism.
Celastrus suppresses the progression of autoimmune arthritis in rats by reducing cellular migration as well as production of pro-inflammatory cytokines and chemokines (116.11)
Authors: Astry Brian , Venkatesha Shivaprasad , Nanjundaiah Siddaraju , Yu Hua , Tong Li , Moudgil Kamal
Published: January 2023

Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disease that affects the synovial joints. The target organ pathology in arthritis is primarily a result of inflammation caused by T cells and other leukocytes accumulating within the joints. These leukocytes produce pro-inflammatory cytokines and chemokines. The prolonged use of conventional anti-inflammatory drugs by RA patients is associated with severe adverse effects. Therefore, the use of natural plant products is on the rise. However, the mechanism of action of such products is poorly defined. Using the rat adjuvant arthritis model of RA, we assessed whether Celastrus aculeatus Merril (Celastrus), a traditional Chinese medicine, can limit the disease, and also examined the mechanisms underlying its anti-arthritic activity. We tested the draining lymph node cells and synovial-infiltrating cells (SIC) of Celastrus-treated versus control rats using flow cytometry and assays for cytokines and chemokines. Arthritic Lewis rats treated with Celastrus had reduced inflammation and pathological damage in the joints compared to controls. Further, Celastrus treatment reduced the frequency of Th17 in the pathogenic cellular infiltrate within the joint, limited the Th17 cell differentiation in lymphoid tissues, and decreased the production of pro-inflammatory cytokines and chemokines (IL-17, IL-6, IL-1β, and RANTES) by the SIC. We suggest that Celastrus be further evaluated as a complementary therapy for RA.
Tregitope mechanism of action and applications for tolerance induction. (116.8)
Authors: Cousens Leslie , McClaine Elizabeth , Levitz Lauren , Tassone Ryan , Messitt Timothy , Martin William , De Groot Anne
Published: January 2023

Tregitopes are T cell epitopes naturally located in immunoglobulins that bind to multiple MHC Class II alleles and induce regulatory T cell (Treg) responses. Harnessing tolerogenic effects of Tregitopes provides a novel tool to suppress unwanted immune responses and maintain antigen-specific tolerance, thus changing treatment paradigms in autoimmunity. We have now demonstrated that APCs present Tregitopes to natural (n) Treg, engage feedback mechanisms promoting a tolerogenic APC phenotype, induce Treg expansion, and modulate antigen-specific effector T cell responses. Proportions of APC expressing MHC II, CD80, and CD86 are suppressed, consistent with reported effects of IVIG (Bayry et al. Blood, 2003, 101:758) and of the IgG-derived peptide hCDR1 (Sela et al. Immunology, 2009, 128:395). Moreover, we observed significant increases in proportions of IL-10-producing CD4+CD25+ FoxP3-expressing nTreg in the presence of Tregitopes. These studies are an exciting first step towards understanding the basic mechanism of Tregitope tolerance induction that we propose to be as follows: 1) APC present Tregitopes to nTreg, 2) nTreg are activated to proliferate and produce IL-10, 3) nTreg provide tolerogenic feedback signals to APC, modulating the APC phenotype, and 4) nTreg and tolerogenic APC together suppress antigen-specific T cell responses. These data suggest a role for Tregitopes in the mechanism of action for IVIG, and provide insight critical for future therapeutic applications.
Inflammatory monocyte recruitment is regulated by IL-23 during systemic bacterial infection (117.1)
Authors: Indramohan Mohanalaxmi , Sieve Amy , Break Timothy , Berg Rance
Published: January 2023

Inflammatory monocytes are innate immune cells that produce TNF-α and NO., and are essential for resistance against various pathogens, including Listeria monocytogenes (LM). IL-23 is an important pro-inflammatory cytokine well known for its role in neutrophil recruitment during multiple infectious and autoimmune diseases. We have previously shown that IL-23 is required for host resistance against LM and for neutrophil recruitment to the liver, but not the spleen, during infection. Despite efficient neutrophil recruitment to the spleen, IL-23p19 knockout (KO) mice have an increased bacterial burden in this organ. Interestingly, specific depletion of neutrophils abrogated the differences in bacterial burden in the livers, but not the spleens of C57BL/6 and IL-23p19 KO mice, suggesting that IL-23 may regulate the recruitment/function of another cell type to the spleen. Accordingly, LM-infected IL-23p19 KO mice had fewer monocytes in the spleen, as well as a reduction in monocyte-recruiting chemokines compared to C57BL/6 mice. This lack of efficient monocyte recruitment resulted in decreased production of monocyte-derived TNF-α and NO., leading to increased bacterial burdens in the spleens of LM-infected IL-23p19 KO mice. Collectively, our data reveal a novel mechanism by which IL-23 provides protection against bacterial infection.
MiR-146a regulated IL-6 expression promotes monocyte proliferation in sepsis patients (117.22)
Authors: Zhou Juhua , Chaudhry Hina , Zhong Yin , Perkins Linda , McGuire Franklin , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Sepsis is a serious medical condition, which is characterized by a systemic inflammatory state induced by a known or suspected infection. Despite extensive basic research and clinical studies, the pathogenesis of sepsis is still poorly understood. In the current study, we found that total peripheral blood mononuclear cell counts increased significantly, however, lymphocytes including CD4+ and CD8+ T cells decreased significantly in sepsis patients. Further studies demonstrated that monocytes increased significantly in sepsis patients. Plasma levels of IL-6, IL-8 and MCP-1 dramatically increased, whereas plasma levels of RANTES significantly decreased in sepsis patients compared with normal controls. MiRNAs such as miR-143, miR-145, miR-146a, miR-181a, miR-182, miR-199a, miR-486 and miR-1308 were dysregulated in sepsis patients. Down-regulation of miR-146a may be responsible for the increased levels of IL-6 expression, and thus may promote monocyte proliferation in sepsis patients. These results suggest that the modulation of IL-6 expression by dysregulated miR-146a in the control of monocyte proliferation may have implications in the pathogenesis and treatment of sepsis.
Natural killer lytic-associated molecule is a component of macrophage phagosomes and contributes to bacterial killing (117.23)
Authors: Lawrence Donald , Kornbluth Jacki
Published: January 2023

Macrophages are a critically important component of the immune system. They are equipped with oxidative and non-oxidative mechanisms to kill pathogens that have been compartmentalized into phagosomes following phagocytosis. Ubiquitin, a small protein modifier that regulates protein degradation, has been found to play a role in macrophage bacterial killing. Natural killer lytic-associated molecule (NKLAM) is an E3 ubiquitin ligase expressed in macrophages and natural killer cells. We have shown that NKLAM expression in macrophages is enhanced by Toll-like receptor agonists and pro-inflammatory cytokines. Using bone marrow-derived macrophages from wild type (WT) and NKLAM-deficient mice we found the levels of phagocytosis and phagosome acidification to be similar; however, killing of E. coli by NKLAM-deficient macrophages is less than by WT macrophages. Using confocal immunofluorescence microscopy, we found that NKLAM is colocalized with ingested bacteria. In assays using IgG-opsonized latex beads as targets, we demonstrated that NKLAM is translocated to the phagosome early in the maturation process. In addition, the presence of NKLAM in the phagosome temporally coincides with increased levels of ubiquitin. Collectively, our data demonstrate that NKLAM is a novel component of macrophage phagosomes and that NKLAM is involved in macrophage bactericidal functions.
Type I interferon induces necroptosis in macrophages during Salmonella Typhimurium infection (117.8)
Authors: Sad Subash , Robinson Nirmal , Mccomb Scott , Krishnan Lakshmi
Published: January 2023

Salmonella typhimurium (ST) is a virulent pathogen that induces rapid host-fatality. We observed that host-survival was enhanced in the absence of IFN-I signalling. Cell-transfer experiments indicated that increased survival of IFN-IR-deficient mice was attributed to efficient control of ST by macrophages. While there was no impairment in inflammasome activation in IFN-IR-deficient macrophages they were highly resistant to ST-induced cell-death. Specific inhibition of receptor interacting protein kinase-1 (RIP-1), or knock-down of RIP-3 prevented the death of wild-type macrophages, implicating necroptosis as a mechanism of cell-death. Furthermore, RIP-3-deficient mice, lacking necroptosis, displayed reduced macrophage-death and enhanced control of ST in vivo. Finally, IFN-I signalling directly induced RIP-1 phosphorylation in macrophages. Thus, ST exploits IFN-I-signalling to activate necroptosis of macrophages and promote immune-evasion.
Eosinophil-mediated lung host defense against Pneumocystis murina (118.6)
Authors: Nelson Michael , Burg Ashley , Metz Allison , Werner Jessica , Dunaway Chad , Steele Chad
Published: January 2023

We have recently reported that Src TKO mice, which demonstrate augmented lung clearance of P. murina (PM) (Infect Immun, May 2009), have enhanced expression of alternative macrophage activation (M2a) markers and the M2a-associated chemokines CCL17 and CCL22 (J Immunol, February 2011). As CCL17 and CCL22 often drive the recruitment of eosinophils, we questioned their role in PM lung defense. PM infection resulted in significantly increased expression of eosinophil peroxidase (Epx) mRNA, which was further enhanced in mice with greater resistance to PM lung infection. We further observed PM-induced mRNA expression of Prg2, but not Ear1 or Ear2, in the lungs. GATA1 deficient mice, which have a selective deficiency in eosinophils, demonstrated impaired PM lung clearance. In turn, Epx and Prg2 mRNA expression was significantly lower in GATA1 deficient mice. Finally, bone marrow-derived eosinophils exhibited the capacity to kill PM in vitro. Collectively, our work supports a role for eosinophils in lung host defense against Pneumocystis.
Tolerization of diabetogenic CD4+ T cells by intranasal treatment with CTA1R7K-DD containing specific peptide through the induction of FoxP3+ Treg cells (119.10)
Authors: Cardell Susanna , Fahlén-Yrlid Linda , Mark Linda , Löfbom Linda , Lycke Nils
Published: January 2023

Type I diabetes (T1D) results from immune destruction of insulin producing β-cells in the pancreas islets. Diabetogenic CD4+ T cells are key cells in the autoimmune process. To achieve tolerization of diabetogenic CD4+ T cells would therefore be an important advancement in the development of treatments of T1D. We previously described that a mutated (R7K), enzyme killed, form of the cholera toxin A1 subunit based adjuvant CTA1-DD induces specific tolerance rather than enhancement of immunity. Intranasal (i n) treatment with CTA1R7K-DD containing a type II collagen peptide reduced in vitro recall responses to the peptide, and moreover, ameliorated collagen induced arthritis in mice. Here, we use CTA1R7K-DD to investigate tolerization of diabetogenic CD4+ T cells of the non-obese diabetic (NOD) mouse, exploring diabetogenic TCR transgenic BDC2.5 CD4+ T cells. I n treatment of BDC2.5 NOD mice with CTA1R7K-DD containing a peptide specific for the BDC2.5 TCR reduced proliferation and IFN-γ production to in vitro peptide stimulation. Transfer of CD4+ BDC2.5 T cells to NOD.scid mice results in T1D development in 80-100% of recipient mice. In contrast, recipients of cells from BDC2.5 NOD mice treated with the CTA1R7K-DD peptide construct remained healthy. The i n treatment resulted in systemic increase in the frequency of CD4+ BDC2.5 transgenic T cells expressing FoxP3, suggesting that CTA1R7K-peptide-DD induces specific CD4+ Treg cells preventing T1D development.
Targeting the immunoproteasome with the second generation proteasome inhibitor ONX 0914: effects on IFN-alpha-induced monocyte-derived dendritic cells (119.4)
Authors: Verbrugge Sue Ellen , Emal Diba , Al Marjon , Lougheed Sinead , Chan Elena , Kirk Christopher , Dijkmans Ben , Scheper Rik , de Gruijl Tanja
Published: January 2023

Proteasome inhibitors (PI) have been proposed as therapeutic agents for autoimmune diseases. Bortezomib (BTZ) is the first registered PI targeting both the constitutive and the immunoproteasome. However, given reported toxicities, its suitability for long term treatment of chronic inflammation should be exercised with caution. Conceptually, ONX 0914 (ONX), a second-generation PI that specifically targets the immunoproteasome, may cause fewer unwanted side effects. High circulating IFN-alpha levels in autoimmune patients have been linked to systemic dendritic cell (DC) maturation, which may support chronic inflammation. In this study we compared the ability of ONX vs BTZ to interfere with IFN-DC development and function. While at non-toxic concentrations ONX (100 nM) and BTZ (5 nM) hampered differentiation of both IFN-DC and the more conventional IL-4-DC from monocytes (based on loss of CD1a expression), IFN-DC were more profoundly affected by proteasome inhibition; 65% to 25% vs 65% to <5% (median, n=8), respectively. ONX and BTZ equally hampered maturation of IFN-DC (25-30% reduction in CD83 expression) and suppressed (up to 2-fold) their ability to induce allogeneic T-cell proliferation. Finally, release of IL-6 by IFN-DC, but not by IL-4 DC, was suppressed 2-3 fold by both ONX and BTZ. Collectively, these observations suggest that ONX may be equally effective as BTZ in inhibiting inadvertent type-1 IFN-mediated DC activation during chronic inflammation.
IMO-8400, a novel TLR7, TLR8 and TLR9 antagonist, inhibits disease development in mouse models of psoriasis (119.8)
Authors: Jiang Weiwen , Zhu Fugang , Tang Jimmy , Kandimalla Ekambar , La Monica Nicola , Agrawal Sudhir
Published: January 2023

Toll-like receptors (TLR) are involved in the pathogenesis of numerous autoimmune diseases, including psoriasis. The role of TLR7 and TLR9 in psoriasis is well established and TLR8 may also contribute to this disease. Blocking the activation of these receptors through a TLR antagonist represents a novel approach to the treatment of psoriasis. We have developed IMO-8400, a synthetic DNA compound that inhibits TLR7-, TLR8-, and TLR9-mediated immune responses in cell-based assays. Additionally, following subcutaneous administration in non-human primates, it suppresses ex-vivo immune responses mediated through these receptors. IMO-8400 was evaluated in preclinical mouse models of psoriasis, induced by IL-23 cytokine or antimicrobial peptide LL-37. Psoriasis-like skin lesions were induced in the ear of C57BL/6 mice by intradermal injection of IL-23 or LL37. Injected mice were treated with subcutaneous administration of IMO-8400. Mice were euthanized at end of study and tissue samples were taken for histological examination. Increase in ear thickness was inhibited in a dose-dependent manner by treatment with IMO-8400. Reduction in epidermal hyperplasia and infiltrating leukocytes was observed in sample tissues. Treatment with IMO-8400 was associated with elevation of serum IL-10 levels. IMO-8400 can effectively suppress lesion development in these psoriasis models. IMO-8400 has potential as a novel agent for the treatment of psoriasis and other autoimmune diseases.
A Dual PIM 1/3 Kinase Inhibitor Demonstrates Efficacy in Murine Models of Lupus and Multiple Sclerosis (119.9)
Authors: Robinson John , Pheneger Jed , Patrice Lee , Wright Dale , Brown Suzy , Sullivan Francis , Davis Gregg
Published: January 2023

PIM kinases are a family of 3 intracellular serine/threonine kinases, and have been shown to be uniquely regulated at the level of expression rather than by upstream phosphorylation. The PIMs are STAT-induced proteins and function as a key signaling node downstream of multiple cytokine, growth factor and oncogene pathways. Here we report the in vivo efficacy in mice of a small molecule PIM kinase inhibitor, AR317, in NZB/NZW Lupus, MOG EAE, and relapsing remitting EAE models. AR317 is a selective and potent inhibitor of PIM-1 and PIM-3 kinases in both the enzyme (IC50 = 0.2 nM PIM-1 and 0.5 nM PIM-3) and cellular setting (EC50 = 21 nM). Administered orally twice daily at 3, 10, and 30 mg/kg to NZB/NZW mice, AR317 afforded dose-responsive improvements in proteinuria, with normalization to base line at 30 mg/kg. Improvements in disease indices were also observed including reductions in spleen and kidney weights, increase in hematocrit, decreases in lymphocyte and neutrophil counts. In a mouse SJL MOG (35-55) EAE model, AR317 (70 mg/kg, BID) produced a significant reduction in ataxia over the course of the study. When dosed in the SJL/J PLP(139-151) mouse model of relapsing remitting EAE, AR317 exhibited marked efficacy vs. ataxia, with reductions in cumulative relapse scores at both 30 mg/kg and 90 mg/kg. These data support the hypothesis that a small molecule PIM 1/3 kinase inhibitor may provide efficacy for patients suffering from lupus or multiple sclerosis.
β-Arrestin-1 deficiency protects mice from experimental colitis (120.16)
Authors: Lee Taehyung , Lee Eunhee , Raehtz Sandi , McCabe Laura R. , Parameswaran Narayanan
Published: January 2023

β-arrestin-1 (β-arr-1) is an intracellular scaffolding protein demonstrated to regulate a number of different receptor-mediated signaling pathways, including Toll-like receptors in immune cells. Based on our recent studies in the regulation of TLR signaling by β-arr-1, we tested the hypothesis that β-arr-1 plays an important role in the pathogenesis of inflammatory bowel disease (IBD). Our results demonstrate that β-arr-1 knockout (β-arr-1-/-) mice are protected from experimental colitis compared to WT mice as determined by body weight loss, clinical score, and colon length. Consistent with the phenotypic consequences, histological examination and myeloperoxidase activity in the colon revealed attenuated inflammatory cell infiltration in the β-arr-1-/-mice compared to WT mice. To further determine the mechanisms, we examined various cytokines implicated in the pathogenesis of colitis. Our results demonstrate that β-arr-1-/- mice are deficient in expression and secretion of IL-6 in the colon compared to the WT mice. Taken together, these results demonstrate that β-arr-1-dependent signaling plays an important role in the pathogenesis of colitis likely via IL-6 production and therefore provides a potential pharmacological target in the treatment of IBD.
The neuropeptides α-MSH and NPY differentially modulate phagocytosis by macrophages (120.24)
Authors: Phan Toan , Mylvaganam Hari , Taylor Andrew
Published: January 2023

The ocular microenvironment tightly regulates immunity to prevent inflammation. These anti-inflammatory mechanisms are partly mediated by immunomodulating neuropeptides. We previously found that alpha-melanocyte stimulating hormone (α-MSH) and Neuropeptide Y (NPY) together induce activation of myeloid suppressor-like macrophages. In this study, we examined the possibility that α-MSH and NPY also modulate phagocytosis by macrophages. The monocytic cell line RAW 246.7 was treated with α-MSH or NPY at 1 ng/ml each, a concentration produced by retinal cells in culture. The treated cells were feed florescent bioparticles of Gram(-) E. Coli or Gram(+) S. Aureus with or without opsonization. Also, tested was the formation of phagolysosomes using pH sensitive florescent E. Coli or S. Aureus bioparticles. Phagocytic activity was assayed by flow cytometry. The α-MSH and NPY treated macrophages were significantly suppressed in their capacity to phagocytize unopsonized E. coli; however, suppression of S. Aureus phagocytosis was limited to NPY treated macrophages. In addition, α-MSH/NPY co-treatment suppressed both phagocytosis and phagolysosome formation in the macrophages. There was no change in phagocytic activity if the bioparticles were opsonized with antibody or complement. Therefore, as α-MSH and NPY together induce suppressor macrophages within the ocular microenvironment, they also modulate in a stimulus-dependent manner phagocytic signals within the macrophages.
Signaling through the P2X7 receptor for ATP induces psoriasis-like inflammation in human skin (120.28)
Authors: Mathers Alicia , Killeen Meaghan , Ferris Laura , Falo Louis
Published: January 2023

Current therapeutics for psoriasis includes the use of systemic acting immuno-suppressive drugs; however, long-term effects of these treatments remain a concern. A better understanding of the mechanism(s) involved in psoriasis pathogenesis is highly relevant for developing refined therapeutics. However, the immunological processes that perpetuate psoriatic lesions, particularly factors that stimulate DCs to bias autoimmune Th17 responses, are not well known. It is likely that alarmins, such as ATP, play a pivotal role in the pathogenesis by positive-feedback inflammatory mechanisms that break immunological self-tolerance and perpetuate immune responses. We have demonstrated the presence of P2X7R, an ATP receptor, in lesional and non-lesional psoriatic skin, suggesting a principal difference in skin of psoriasis patients and not just a change due to inflammation. Mechanistically, utilizing human cutaneous models, we determined that signaling via P2X7R initiates psoriasis-like inflammation in skin by mechanisms that include innate immunity, DC17 differentiation, and the induction of Th17 biased responses. Furthermore, our results define a prospective role for P2X7R signaling in perpetuating psoriasis lesions by stimulating the mir-21/VEGF pathway. In conclusion, we have begun to describe a potential mechanism of psoriasis lesional formation in humans, which will contribute to understanding the pathogenesis of psoriasis and lead to the development of refined therapeutics.
TIM-1 mediates the induction of ozone-induced airway hyperreactivity through the recognition of apoptotic cells in the lung (120.33)
Authors: Kim Hye Young , Lee HyunHee , Chang Ya-Jen , Chaung Ya-Ting , Shore Stephanie , Freeman Gordon , DeKruyff Rosemarie , Umetsu Dale
Published: January 2023

Asthma is a heterogeneous inflammatory disease of the lungs that has multiple triggers, including air pollution. The mechanisms however, by which non-allergic triggers such as air pollution cause asthma are poorly understood. In a model of air pollution-induced asthma, we found that chronic exposure to ozone induced airway hyperreactivity (AHR), a cardinal feature of asthma, independently of adaptive immunity. The AHR induced by ozone was however, dependent on TIM-1, an important susceptibility gene for asthma and a receptor for phosphatidylserine (PtdSer), a marker of apoptotic cells. Moreover, ozone exposure caused apoptosis in airway epithelial cells, which triggerred AHR by activating TIM-1 expressing natural killer T (NKT) cells. These results demonstrate an important role for TIM-1 in asthma, in which AHR results from the activation of NKT cells sensing damaged/apoptotic airway epithelial cells through TIM-1. We thus define a novel innate pathway to experimental asthma that involves apoptosis and TIM-1, and that links airway injury to inflammation and AHR.
MyD88 Expression on Villin Inhibits Obesity in High Fat Diet Mice (120.5)
Authors: Chao Chen , Xiang Yufei , Wang Yipeng , Peng Jian , Hu Youjia , Tai Ningwen , Hoshi Namiko , Zhou Zhiguang , Medzhitov Ruslan , Wen Li
Published: January 2023

To investigate whether MyD88 in the gut epithelial brush border plays a role in obesity and metabolic syndrome, we studied a transgenic mouse line expressing MyD88 only in villi (VillinMyD88+/+MyD88-/- mice). In this study, we fed young male wild type B6 (n=8), MyD88-/-B6 (n=6) and VillinMyD88+/+MyD88-/-B6 mice (n=13) with high fat diet (HFD) for 16 weeks. We investigated the metabolic, immunological and gut bacteria profiles of these mice. We found that VillinMyD88+/+MyD88-/-B6 mice had lower body weight, net weight gain, food consumption, total body fat mass, and white adipose tissue weight compared to B6 and/or MyD88-/-B6 mice (P<0.05). Glucose and insulin tolerance tests showed that VillinMyD88+/+MyD88-/-B6 mice were more insulin sensitive than B6 and MyD88-/- B6 mice (P<0.05). Interestingly, white adipose tissue in VillinMyD88+/+MyD88-/-B6 mice harbored fewer B cells, Th17 cells and IL-6+ cells than B6 and/or MyD88-/-B6 mice (P<0.05). Moreover, the number of CD103+CD8+ cells were lower in spleen, mesenteric lymph node, and Peyer Patches of VillinMyD88+/+MyD88-/- B6 mice (P<0.05) compared with B6 and MyD88-/-B6 mice. Gut flora study showed that B6 mice had more Lactobacillus, while MyD88-/-B6 and VillinMyD88+/+MyD88-/-B6 mice had more Enterococcus. However, VillinMyD88+/+MyD88-/-B6 mice had a higher ratio of Gram+/Gram- than MyD88-/-B6 mice in faeces. Our data revealed a novel role of MyD88 in gut epithelial brush border, which affects obesity and metabolism.
An IKKβ-IL-17 axis fine-tunes Ab responses to sublingual immunization (120.7)
Authors: Jee Junbae , Bonnegarde-Bernard Astrid , Iwakura Yoichiro , Karin Michael , Cormet-Boyaka Estelle , Boyaka Prosper
Published: January 2023

The nature of immune cells recruited at the sites of antigen exposure and the cytokine microenvironment play a key role at shaping immune responses. Using a model sublingual vaccine with Bacillus anthracis edema toxin as an adjuvant, we show that alteration of IKKβ signaling in myeloid cells (IKKβΔMye) enhanced the magnitude of Ab and T cell responses, compared to control mice. While this model vaccine failed to induce S-IgA Abs in control mice, significant S-IgA Ab responses were induced in mucosal secretions of IKKβΔMye mice. We also established that the sublingual vaccine recruited lower numbers of CD11b+F4/80+Gr1high and CD11b+F4/80-Gr1high, but higher number of CD11b+F4/80+Gr1- cells in the sublingual tissues of IKKβΔMye mice. In contrast, higher numbers of CD11b+F4/80+Gr1high and CD11b+F4/80-Gr1high, but lower number of CD11b+F4/80+Gr1- cells were recruited in the sublingual tissues of control and IL-17A KO mice. Lack of IL-17A signaling did not affect the magnitude of Ab responses to the sublingual vaccine. However, the profile of B cell epitope responses was affected in IL-17A KO mice. Furthermore, CD11b+ cells isolated from IKKβΔMye mice assisted B cell-differentiation and induced production of IgA Abs by IL-17Rhigh B lymphocytes in vitro. Our results support the existence of an IKKβ-IL-17 axis for regulation of mucosal immunity and identify IKKβ signaling as a potential target for improving induction of mucosal immunity by sublingual vaccines.
Full-length Interleukin-33 modulates the effects of Interleukin-4 in the lungs in vivo (120.8)
Authors: Kopach Pavel , Lockatell Virginia , Kang Phillip , Pickering Edward , Atamas Sergei , Luzina Irina
Published: January 2023

Previous reports have been inconsistent on whether Interleukin (IL)-33 necessarily promotes Th2 effects in vivo. This diversity of findings may be due to post-synthetic processing of IL-33. We compared the in vivo effects of full-length mouse (flm) and mature mouse (mm) IL-33 utilizing replication-deficient adenovirus (AdV)-mediated gene delivery. mmIL-33 promoted pulmonary eosinophilia, neutrophilia, and significant elevations in IL-4, IL-5, and IL-13 compared to AdV-NULL-infected controls (p < 0.05 in all cases), whereas flmIL-33 had no such effects. A possibility was considered that flmIL-33 may modulate the effects of a prototypic Th2 cytokine, IL-4. The effects of AdV-mediated gene delivery of IL-4 were compared with the effects of combined flmIL-33 and mIL-4. In the latter case, a bicistronic AdV construct encoding flmIL-33 and mIL-4 was used to ensure co-localization of expression of both cytokines. On day 7 after instillation of the viral particles, IL-4-expressing mice had elevated eosinophils and neutrophils in their BAL, and animals expressing both flmIL-33 and IL-4 had yet significantly more BAL eosinophils and neutrophils compared with IL-4-expressing mice (p < 0.05). Similar potentiating effects of flmIL-33 were observed on IL-4-driven increases in pulmonary cytokines TNF-α, IL-5, IL-13, MCP-1, and in goblet cell hyperplasia. These results suggest that although full-length IL-33 has no independent pro-Th2 effect on the lung, it potentiates such effects of IL-4.