bluuebunny/crossref_metadata_2025 huggingface dataset:
Poxviral infection elicits human neutralizing antibodies recognizing diverse epitopes of the major vaccinia virus surface protein D8 (VIR4P.1016)
Authors: Gilchuk Iuliia , Blum David , Fritz Gloria , Singh Vidisha , Kose Nurgun , Yu Yingchun , Sapparapu Gopal , Matho M. , Zajonc Dirk , Xiang Y. , Hammarlund Erika , Slifka Mark , Crowe James
Published: January 2023

Immunization with vaccinia virus (VACV) confers cross-protective immunity to variola, the causative agent of smallpox, and other clinically important poxviral species, such as cowpox and monkeypox viruses. Vaccine-mediated protection has been correlated with elicitation of broad neutralizing antibody (Ab) responses, however the specificity of human neutralizing antibodies to poxviruses is poorly understood. We used a highly-optimized human hybridoma technology to generate large panels of anti-VACV monoclonal Abs (mAbs) from vaccinia-immunized subjects or from subjects who had naturally acquired monkeypox infection. Immunologic characterization and DNA sequencing of the panel revealed 16 individual mAbs that recognized the immunodominant surface protein antigen D8. Several of anti-D8 mAbs possessed neutralizing activity against VACV, cowpox and monkeypox viruses. These Abs exhibited mainly a complement-dependent pattern of neutralization and they formed at least four cross-blocking groups, suggesting the existence of several non-overlapping neutralizing epitopes for D8. The results suggest that D8 is an important target for human neutralizing Abs generated following poxviral infection or vaccination, and suggest the existence of novel epitopes targeted by anti-D8 Abs that contribute to cross-protective immunity following smallpox vaccination.
Despite large clonal expansions of cytotoxic T cells with age and chronic CMV exposure, the underlying repertoire appears completely normal, suggesting that holes in the T cell repertoire are not responsible for immunosenesence (VIR4P.1012)
Authors: Mukherjee Rithun , Lindau Paul , Desmarais Cindy , DeGloria Jeanne , Utsugi Heidi , Warren Edus , Riddell Stanley , Makar Karen , Turtle Cameron , Robins Harlan
Published: January 2023

Age is thought to restrict the cytotoxic immune repertoire, both in terms of number and diversity of naïve T cells available for responding to novel pathogens and vaccinogens. Studies have shown that CMV-induced chronic stimulation exacerbates this immune restriction. CMV infection leads to CD8 T cell clonal expansions (TCEs), bearing classical memory as well as late-differentiated memory phenotypes. Such TCEs become huge in the elderly, where they have been held responsible for constriction of the remainder of the repertoire. In turn, this has been linked with the CMV-associated immune risk profile and increased mortality in the elderly, although it is not known how TCEs affect immune competence. Here we address the premise that immune competence with age could be compromised because of repertoire restriction. We do so by describing the structure of the elderly repertoire at an unprecedented resolution in terms of both clonal origin and broad CD8 T cell phenotypes. We then proceed to exhaustively identify CMV-specific clones. This presents us with a close look at the rest of the repertoire, which does not respond to CMV. We compare this with the full repertoire in CMV- individuals. Our observations suggest that CMV-specific TCEs are restricted to components of the repertoire that expand without affecting the absolute size or diversity elsewhere in the repertoire, so “holes” are unlikely to develop in the T cell repertoire.
Mechanisms of enhanced TRAIL, CD86, and CD40 expression by plasmacytoid dendritic cells from HIV-infected subjects (VIR5P.1027)
Authors: Serei Virian , Dai Jihong , Hodder Sally , Fitzgerald-Bocarsly Patricia
Published: January 2023

Plasmacytoid dendritic cells (pDC) are critical in viral immunity via their production of IFN-α and presentation of antigen. In HIV+ subjects, pDC decline impairs immune defense, while chronic in vivo stimulation of pDC contributes to immune activation. Here, we aimed to further explore pDC dysfunction in HIV+ subjects. We confirmed that pDC from HIV+ subjects have increased TRAIL, CD86, and CD40 expression compared to healthy donors. pDC from both HIV+ and healthy subjects upregulated TRAIL, CD86, and CD40 in response to overnight HIV- and HSV-stimulation. However, pDC from HIV+ subjects with CD4 >200 showed increased TRAIL, CD86, and CD40 upregulation compared to healthy donors and HIV+ subjects with CD4 <200. pDC from healthy donors were then used to explore how expression of these molecules occurs. Inhibition of endosomal acidification blocked TRAIL, CD86, and CD40 in virus-stimulated pDC. TLR7 inhibition in HIV-stimulated pDC partially inhibited this expression. While TLR9 blockage also inhibited CD40 expression by HSV-stimulated pDC, there was no effect on CD86 and TRAIL. Pro-inflammatory cytokines IL-6, TNF-α, and IFN-α were able to enhance TRAIL expression by HIV-stimulated pDC. Despite upregulation of CD86, HIV- stimulated pDC from healthy donors were poor inducers of allogeneic CD4 T cell expansion. We conclude that pro-inflammatory cytokines, along with TLR signaling, “prime” pDC in HIV-infected individuals to express TRAIL, CD86, and CD40.
Latent viral infection alters the immune architecture of the lung (VIR5P.1030)
Authors: Dwivedi Varun , Trgovcich Joanne , Mansfield Sara , Cook Charles
Published: January 2023

Human cytomegalovirus (HCMV) is ubiquitous virus infecting most humans and developing persistent latent infections. HCMV reactivation occurs in ~33% of immune-competent patients during critical illness associated with doubled risk of mortality. In many persistent infections Myeloid derived suppressor cells (MDSC) cause T cell dysfunction, creating an immunosuppressive microenvironment. We hypothesized that latent CMV infection induces MDSC, perhaps contributing the impairment of host resolution of infection and thus latency. BALB/c mice were infected with 1×106 PFU of Smith strain murine CMV and lung mononuclear cells (LMNC) were cultured ex vivo or stained for surface markers and analyzed by ELISA or FACS. During latency, we observed a change in frequency (million/gm of lung tissue) of cell subsets such as marked reduction of CD4+T and B cells, increased CD8+T cells and significant increase in NK cells compared to naïve mice. Interestingly, the percentage of MDSC was unchanged at 2 weeks, but was significantly reduced at12 weeks post infection (p.i.). Both IL-6 and TNF-α were elevated in unstimulated or LPS, ConA stimulated LMNC at 2 and 12 weeks p.i. conferring the risk of lung pathology under critical illness. Our findings suggest that during the transition from acute to latent infection, persistent pro-inflammatory lung immune environment is associated with reduced MDSC. Longitudinal studies are underway to explore the role of MDSC in lungs of latently infected mice.
Dysregulated host inflammatory response in rats with fatal encephalitic Rift Valley Fever (VIR5P.1021)
Authors: Hartman Amy , Caroline Amy , Bethel Laura , Powell Diana , Oury Tim , Reed Douglas
Published: January 2023

Rift Valley Fever virus (RVFV) is a zoonotic arbovirus capable of causing widespread outbreaks with severe morbidity, mortality, and socioeconomic consequences. Most patients develop an acute febrile disease but recover, while a small subset of infected humans develop more severe disease including hemorrhagic manifestations, meningoencephilitis, or retinitis resulting in blindness. Inbred rats have been shown to be a good model of severe human RVF. Lewis rats are highly resistant to subcutaneous inoculation with RVFV, but develop uniformly fatal encephalitis after exposure to low doses of RVFV by inhalation. Elevated levels of M-CSF and MCP-1 are seen in the blood on day 4 post-exposure concurrent with granulocytosis and thrombocytopenia that continues until death. IL-1β and GRO/KC, the rat homolog of IL-8, peaked slightly on day 4 p.i., decreased briefly, and were highly elevated in the blood of moribund rats. In the brain, many pro-inflammatory cytokine levels, including IL-1α, IFN-γ, and IL-18, were found to be greatly elevated at day 5 post-exposure, which coincides with the earliest detection of virus in the brain. Pathological evaluation of brain tissue indicates an influx of lymphocytes and neutrophils that corresponds with the onset of neurological signs and severe disease in the Lewis rats. These data suggest that the host immunological response may be contributing to the pathogenesis of RVFV in the brain and the outcome of infection.
Human cytomegalovirus microRNA miR-UL148D facilitates HCMV latency (VIR5P.1024)
Authors: Liu Yuan , Pan Chaoyun , Li Donghai , Liu Fenyong , Zen Ke
Published: January 2023

Human cytomegalovirus (HCMV), infects a majority of population and can undergo life-long, latent infection, is a leading cause of opportunistic and congenital disease. HCMV can encode 17 microRNAs (miRNAs) but the roles of these viral miRNAs during viral infection are unclear. Here, we infected human bone marrow and CD34+ hematopoietic progenitor Kasumi-3 cells with HCMV (strain NR-1) and determined the kinetics of cellular levels of all hcmv miRNAs by qRT-PCR. We found that, different from most other hcmv miRNAs which had high level at early days of infection, hcmv-miR-UL148D was highly expressed at the latent infection (10days or longer). Bioinformatics and experimental validation showed that hcmv-miR-UL148D targets the 3’UTR of host cell immediately-early response gene 5 (IER5). Functional assay indicated that downregulation of IER5 by hcmv-miR-UL148D transcriptionally upregulated CDC25B, leading to lower number of Kasumi-3 cells in G2/M phase. As G2/M phase is a cell cycle of large viral replication, decrease the number of cells in G2/M phase by hcmv-miR-UL148D would suppress viral replication and facilitate hcmv entering in latent infection. In contrast, NR1-Δhcmv-miR-UL148D, an hcmv mutant deleting hcmv-miR-UL148D, was lack of ability to target IER5, increase CDC25B, control the cell cycle of host cells, and achieve latent infection. Our results collectively demonstrate that hcmv-miR-UL148D facilitates hcmv latency through modulating host cell IER5-CDC25B axis.
Identification of a specific gene signature in human Th1/17 cells (BA13P.126)
Authors: Duhen Thomas , Ni Chester , Campbell Daniel
Published: January 2023

T cells that co-express the chemokine receptors CCR6 and CXCR3 and produce IL-17, IFN-γ, GM-CSF and low IL-10, also known as Th1/17 cells, have been proposed to be highly pathogenic in several autoimmune disorders. To better characterize these cells and examine their relationship with Th1 and Th17 cells, we compared the whole transcriptome of the aforementioned T cell subsets by RNAseq directly ex vivo. Using this approach, we identified groups of genes shared either with Th1 or Th17 cells. The Th1 signature included genes such as TBX21, IL12RB2, GZMK, PRF1 and CCL5, whereas the Th17 signature contained well-established Th17-associated genes (i.e. RORC and KLRB1) as well as those not previously associated with Th17 cells (i.e. CTSH and PTPN13). Interestingly, we also found genes specifically expressed by Th1/17 cells, among which are IL4i1, LTK and ABCB1. The latter, also known as MDR1, encodes for a transmembrane transporter playing a major role in the efflux of drugs and endogenous metabolites across the cell membrane. We confirmed its unique expression on Th1/17 cells using a rhodamine efflux assay. Moreover, MDR1+ Th1/17 cells displayed a highly pro-inflammatory cytokine profile. Our data reinforce the notion that Th1/17 cells are a heterogeneous population of cells combining Th1 and Th17 phenotypic and functional characteristics but also that these cells have unique properties that could be targeted as new strategies to combat autoimmune diseases.
Regulatory T cells in B cell-deficient mice differ from those in WT mice in function and expression of cell surface markers (BA13P.119)
Authors: Ellis Jason , Braley-Mullen Helen
Published: January 2023

NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) with chronic inflammation of thyroids by T and B cells. B cell-deficient (B-/-) mice are resistant to SAT unless Treg are transiently depleted. To determine if Treg from WT and B-/- mice differ in their ability to suppress effectors from a single source, splenocytes from CD28-/-B-/- mice (effector cells and APC) were cultured with or without sorted Treg from Foxp3-GFP WT or B-/- mice before transfer to mice lacking T cells. Recipients of Treg from B-/- mice had significantly lower SAT severity scores compared to those given Treg from WT mice, indicating that Treg in B-/- and WT mice differ functionally. Treg from WT and B-/- mice also differ in expression of TNF receptor superfamily members CD27, TNFR II p75, and GITR. After transient Treg depletion with anti-CD25 or diphtheria toxin, repopulating Treg in B-/- mice had reduced suppressor function, and expression of CD27, p75 and GITR was like that of WT Treg. If B cells were present during development of B-/- Treg in bone marrow chimeras or added to cultures of B-/- Treg and T effectors, expression of CD27, p75 and GITR by Treg from B-/- mice was comparable to that of WT Treg, and suppressive function was reduced. These results establish for the first time that Treg in WT and B-/- mice differ both functionally and phenotypically, and both are altered when B cells are present during Treg development and during interaction of Treg with T effectors.
Leptin promotesTh17 cell response and exacerbates collagen-induced arthritis (BA13P.120)
Authors: Deng Jun , Lu Liwei
Published: January 2023

Leptin regulates CD4+ T helper cell differentiation and functions. IL-17-producing CD4+ T cells (Th17 cells) play a pivotal role in the pathogenesis of various autoimmune diseases. Elevated leptin levels have been detected in the sera and synovial fluid of patients with rheumatoid arthritis (RA). However, whether and how leptin regulates Th17 cell responsiveness and its potential application in autoimmune arthritis remains elusive. Using immunohistochemistry staining and immunofluorescence microscopy, we found that elevated leptin levels and increased Th17 cells were closely related to disease severity and joint damage in mice with collagen-induced arthritis. Leptin treatment significantly promoted Th17 cell differentiation, upregulated ROR-gamma t expression and increased IL-17 production in culture. Intra-articular injection of leptin to the joint led to earlier onset of arthritis, higher clinical score, and severer joint damage compared to PBS-injected mice. Th17 cell frequencies and total cell number in the draining lymph nodes and joint tissue were elevated upon leptin injection. Moreover, increased Th17 cells infiltrated into the synovium with increased IL-17 levels were detected in the synovial cavity in leptin-injected mice. Together, these data have demonstrated that leptin plays an important role in the exacerbation of autoimmune arthritis via promoting Th17 cell differentiation and increases IL-17 production.
Overexpression of Pbx1-d, a novel splice isoform of Pbx1, changes the portion of follicular CD4 T cell populations and impairs regulatory T cell homeostasis (BA14P.201)
Authors: Choi Seung-Chul , Zeumer Leilani , Morel Laurence
Published: January 2023

We have previously shown that the differential expression of a novel splice isoform, Pbx1-d, of the Pbx1 gene was associated with the production of autoreactive CD4+ T cells in the Sle1a1 lupus susceptibility locus and lupus susceptibility in the B6.Sle1.Sle2.Sle3 mouse model. Pbx1 regulates the transcription of a large number of genes by controlling chromatin accessibility. Pbx1-d is the only known Pbx1 isoform lacking the DNA binding domain, which suggests a dominant negative function, and its expression is more frequently in the CD4+ T cells from lupus patients than in healthy controls. With the hypothesis that Pbx1-d contributes to lupus development by regulating CD4+ T cell effector functions through novel mechanisms, we have generated B6.CD4-Pbx1-d Tg (Pbx1-d Tg) mice, which specifically express Pbx1-d in CD4+ T cells. These Tg mice reproduce the phenotypes of B6.Sle1a1 mice, with increased inflammatory functions of CD4+ T cells and impaired Treg cell homeostasis. Interestingly, the composition of follicular helper and regulatory T cell (Tfh and Tfr) populations, is different in the Pbx1-d Tg mice as well as the B6.Sle1.Sle2.Sle3 mice as compared with wild-type mice. Currently, we are investigating the mechanisms by which Pbx1 regulates Treg homeostasis and follicular T cell differentiation in the Pbx1-d Tg mice, as well as its role in autoimmune pathology in the B6.Sle1.Sle2.Sle3 mice.
Targeted modification of complex N-glycosylation results in a spontaneous CVID-like immunodeficiency with anti-lymphocyte autoimmunity (BA14P.204)
Authors: Ryan Sean , Abbott Derek , Cobb Brian
Published: January 2023

Common variable immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency affecting humans. Although a clear disease etiology remains elusive, a common characteristic of CVID is deficient IgG antibody production to infection or vaccination. Many patients also exhibit symptoms of abnormal T cell function, including an apparent lack of naïve T cells which correlates with clinical severity. We describe here one of the first animal models of spontaneous CVID that incorporates aspects of both humoral and T cell dysfunction. The CVID mice exhibit characteristic deficiencies in IgG responses to both protein and polysaccharide vaccines. Interestingly, the immunodeficiency is associated with decreased T cell activity due to a persistent autoimmune response depleting naïve T cells, which appears to be mechanistically linked to changes in erythrocyte surface N-glycosylation. The N-glycosylation dependent auto-epitopes that emerge on erythrocytes were found to cross-react with naïve T cells independent of changes to the latter’s glycosylation profile. While the use of Ig replacement therapy and antibiotics has reduced the number of infections in CVID, the major inflammatory complications such as lung disease, cancer, and autoimmunity still develop; thus our findings provide potential insight into human CVID beyond hypogammaglobulinemia by addressing the mechanisms that may contribute to the more broad defects in adaptive immunity linked to clinical severity.
Lectin-targeted dendritic cell immunotherapies against experimental autoimmune encephalitis/multiple sclerosis (CAM5P.242)
Authors: Sagar Divya , Foss Catherine , Khan Zafar , Pomper Martin , Jain Pooja
Published: January 2023

The molecular mechanisms of how circulating dendritic cells (DCs) access the CNS across the blood-brain barrier (BBB) remains to be investigated. During neuroinflammation, DCs transmigrate across the BBB in the presence of chemoattractants, particularly CCL2, and initiate immune response. We showed through near-infrared imaging that DC accumulation into lesions within the CNS correlated with severity of inflammation during experimental autoimmune encephalitis (EAE). Actin polymerization on DCs upon CCL2 exposure indicated a migratory phenotype. Transmigration of DCs was paracellular and more efficient than T cells in vitro. To selectively attenuate DC transmigration, we sought to study expression of DC specific C type lectin receptors- CD205 & CD206 (Type I), CD207, -209, -303, CLEC4A, -9A, -10A & -12A (Type II). Different DC subsets- myeloid (mDC), plasmacytoid (pDC) and monocyte-derived (MDDC) had a unique enrichment of lectin receptors and each subset utilized a different receptor to adhere and transmigrate in the presence of CCL2. Phosphoproteomics revealed that these receptors when triggered, activate molecular events known to be involved in actin dynamics, thus substantiating their role in DC transmigration. Importantly, in EAE mice these lectins were validated targets for inhibiting DC transmigration. Thus, the prospect of selectively regulating DC entry into the CNS will open up new treatments against disease pathogenesis and propagation in multiple sclerosis.
miRNA modulation of M1/M2 macrophage phenotype (CAM5P.231)
Authors: Guerau-de-Arellano Mireia , Jablonski Kyle , Amici Stephanie , Gaodet Andrew , Popovich Philip
Published: January 2023

After spinal cord injury (SCI), “M1” (pro-inflammatory) and “M2” (anti-inflammatory) macrophages populate the site of injury. M1 macrophages are neurotoxic and hamper neuroregeneration while M2 macrophages have a pro-regenerative, anti-inflammatory phenotype. Therefore, therapeutic strategies that suppress M1 and enhance M2 macrophages after SCI are actively sought. Micro RNAs (miRNA) are small, non-coding RNAs, approximately 19-24 nucleotides in length, which regulate gene expression at the transcriptional level. miRNA are known to regulate inflammation in autoimmune disease and inflammatory disorders. We therefore hypothesized that miRNA play a key role in differentiation process of macrophages into the pro-inflammatory M1 phenotype. The relative expression of several miRNA was analyzed in vitro in bone marrow-derived macrophages undergoing differentiation into M1 versus M2 phenotypes. We found that M1 macrophages expressed significantly higher levels of miR-155 than M2 macrophages. In addition, miR-155 expression was increased in the injured spinal cord mirroring the increase in M1 macrophages at the site of injury. miR-155 deficiency or silencing resulted in blunted iNOS, IL-1β and TNFα up-regulation in M1 macrophages, among others. In contrast, Arginase-1 expression remained stable in M2 macrophages. These results indicate that miR-155 plays an important role in the differentiation of macrophages into an M1 phenotype.
Visfatin increases the activity of aggrecanases-1 and -2 in human chondrocytes (CCR5P.264)
Authors: Yaykasli Kursat , Dogan Mustafa , Hatipoglu Omer , Yaykasli Emine , Kaya Ertugrul , Ozsahin Mustafa , Uslu Mustafa
Published: January 2023

Visfatin, characterized in 1994 is an adipokine (adipocytokines). It was secreted by not only visceral adipose tissue but also by several tissues. First, visfatin with insulin-mimetic activity has been implicated in the insulin resistance. Finally, it was understand that visfatin involves in several pathological processes like inflammation. The degradation of aggrecan by inflammation induced aggrecanases is a key process for diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA). Aggrecanases-1 and -2, called ADAMTS-4 and -5 respectively are members of a disintegrin and metalloproteinase with thrombospondin motifs enzyme family. The aim of this study was to investigate the potential role of visfatin in aggrecan destruction. Human articular chondrocytes were stimulated with visfatin at 10, 25, 50, 100, 250 and 500 ng/ml doses. Total RNA was isolated and reverse-transcribed by random primer. The aggrecanases-1 and -2 genes expression were analyzed and standardized by β-actin gene expression using real-time polymerase chain reaction. The highest upregulation for aggrecanases-1 and -2 was determined at 500 ng/ml. Obesity is associated with RA by secreting adipocytokines. In addition, the increased serum visfatin level in patients with RA was reported before. Therefore, visfatin become a potential target for aggrecanases upregulation. In conclusion, the high level of visfatin accompanied by obesity has catabolic effect on the aggrecan degradation.
Multi-color fluorescence microscopy detection of chemokine receptors using novel Brilliant Violet™-conjugated monoclonal antibodies (CCR5P.267)
Authors: Zhang Hong , Lundsten Kelly , Ransom John , Yang Xifeng , Nguyen Dzung
Published: January 2023

BioLegend has generated a large library of high affinity antibody clones against mouse and human chemokine receptors that are validated in flow cytometry applications. They are offered as conjugates with the violet-excited Brilliant VioletTM (BVTM) fluorophores. Use of the BVTM fluorophores has greatly increased phenotypic multiplexing capabilities in flow cytometry by increasing the utility of the violet laser. Violet-excited BV421TM antibody conjugates are at least as bright as yellow/green-excited phycoerythrin (PE) and red-excited Alexa Fluor® 647 conjugates. The brightness of violet-excited BV510TM is more on par with the green-excited Alexa Fluor® 488. BV421TM and BV510TM are far brighter and more photostable than the violet-excited Alexa Fluor® 405 and Pacific BlueTM. Here, using a variety of discrete immunologic cell populations bearing different chemokine receptors, BV421TM and BV510TM are shown to also increase multiplexing capabilities and increase resolution in fluorescence microscopy applications. Elsewhere, both BV421TM and BV510TM were excited with a multiphoton laser, indicating their potential utility in deep tissue and live cell imaging applications. The results show that BV421TM- and BV510TM-conjugated monoclonal antibodies are valuable new tools for the scientist wanting to resolve multiple antigens simultaneously with fluorescence microscopy.
CCR6 expression by small intestine lamina propria dendritic cells is critical for surveillance of the luminal contents at the villous epithelial surface (CCR5P.251)
Authors: McDonald Keely , Wheeler Leroy , McDole Jeremiah , Miller Mark , Newberry Rodney
Published: January 2023

The immune system underlying the villous epithelium is continually monitoring its complex environment to appropriately respond with the induction of tolerance or immunity. However little is known about the factors promoting immune surveillance of the luminal contents at this surface. Mice deficient in lymphotoxin beta receptor (LTBR) have a decreased population of dendritic cells (DCs) associated with the villous epithelium. The chemokine CCL20 is expressed by the villous epithelium in a LTBR dependent manner, and CCR6, the only receptor for CCL20, is expressed by the LP-DCs associating with the villous epithelium. Using an in vivo imaging approach, we observed that LP-DC from CCR6 deficient mice were further from the intestinal lumen and flow cytometric analysis revealed they had a decreased population of epithelia associated LP-DCs. Mixed bone marrow transfers confirmed that CCR6 expression by DCs was required for association with the epithelium. In vivo imaging and flow cytometry revealed that LP-DCs from CCR6-/- mice were not impaired at migrating in the lymphatics or populating the mesenteric lymph node. However LP-DCs from CCR6-/- mice were deficient at acquiring epithelial cell proteins and intraluminal ovalbumin, and were deficient in priming immune responses to luminal antigen in the mesenteric lymph nodes. These findings identify an essential role for CCR6 expression by LP-DCs in surveillance of the luminal environment at the villous epithelial surface.
Murine aortic vascular cells produce IL-17C and support inflammation in atherosclerosis. (CCR6P.271)
Authors: Butcher Matthew , Galkina Elena
Published: January 2023

The progression of atherosclerotic lesions involves regular communication between arterial hematopoetic cells and vascular cells through cytokine and chemokine networks. While recent evidence has demonstrated the presence of Interleukin-17A (IL-17A)-producing T cells within atherosclerotic Apolipoprotein E-deficient (Apoe-/-) aortas, the roles of the other IL-17 family members have not been explored. As several recent studies have demonstrated a pro-inflammatory role for epithelial and keratinocyte-derived IL-17C, we sought to examine aortic Il17c expression within atherosclerotic western diet-fed Apoe-/- and Il17a-/-Apoe-/- mice. Il17c mRNA was detected within the aortas of both Apoe-/- and Il17a-/-Apoe-/- mice and several major sources of aortic IL-17C were identified by flow cytometry; including smooth muscle cells (SMCs, 5±0.8%) and CD45-αSMA- cells (5.8±0.8%). Interestingly, elevated aortic IL-17C production was detected within Il17a-/-Apoe-/- mice in comparison to Apoe-/- littermates (6.16±0.24% vs 3.6±0.1%), suggesting an inverse relationship between T cell derived-IL-17A and vascular-IL-17C. To examine the effects of IL-17C and TNFα on aortic chemokine and cytokine production, we explanted whole aortas with IL-17C or IL-17C and TNFα. In this system, IL-17C broadly supported the expression of Ccl2, Ccl7, Cxcl1, Cxcl2, Cxcl5, and Il6. Together, these data suggest that SMC-derived IL-17C supports atherogenic inflammation via cytokine and chemokine production.
Accentuated dichotomy between activation and resting state results in lower basal IL-2R signaling potential in CD4 memory T cells of T1D subjects (HUM7P.284)
Authors: Long S. Alice , Schwedhelm Katharine , Tatum Megan , Cerosaletti Karen , Greenbaum Carla , Buckner Jane
Published: January 2023

The IL-2/IL-2R pathway is clearly implicated in type 1 diabetes (T1D). However, not all of the underlying mechanisms are understood. Known genetic risk alleles in humans account for some, but not all, of the reduction in IL-2R signaling. To determine additional factors, we interrogated signaling in healthy controls and T1D subjects held constant for IL-2R associated risk alleles. Glucose and HbA1c did not correlate with IL-2 response. Nor was reduced IL-2R signaling observed in T2D subjects or CD4 memory cells rested in fresh media. Instead, IL-2 signaling in CD4 memory cells of T1D subjects remained significantly lower than controls even after in vitro activation and proliferation. Together, these data suggest that intrinsic factors contribute to reduced IL-2R signaling in T1D subjects. IL-2R signaling was comparable regardless of CCR7, CD27 or CD62L expression suggesting that differentiation state is not a factor. However, there was an accentuated dichotomy between IL-2 response in activated versus quiescent cells of T1D subjects resulting in a more pronounced reduction in resting cell IL-2 signaling. Thus, in addition to known T1D-associated risk alleles, intrinsic properties related to activation state may modulate levels of basal IL-2R signaling. This is consistent with our finding that reduced IL-2R signaling occurs as early as autoantibody conversion. Therapeutically targeting multiple components of the IL-2 pathway may increase treatment options for T1D.
Serum Melatonin levels in Sjögren patients (HUM7P.310)
Authors: Keskin Goksal , Keskin Dilek , Inal Ali , Ilikci Rahsan
Published: January 2023

OBJECTIVES: Primary sjögren syndrome is a systemic autoimmune disease characterized by immune system abnormalities. It’s unclear how melatonin influences the immune responses. In this study we investigated the levels of serum melatonin in sjögren patients and melatonin’s diurnal rhythm. METHOD: In this study, 47 sjögren patients (35 female, 12 male, mean age 39,7 ± 11,5 years, mean disease duration 8,2 ± 2,7 years) and 28 healthy controls ( 21 female, 7 male, mean age 41,3± 7,7 years) were included. Serum melatonin levels were measured by ELISA, in the morning and night. RESULTS: In the sjögren patients group; the mean serum melatonin levels were 49, 2 ± 12,3 at night and 52,6 ± 5,1 in the morning. In the healthy control group; the mean serum melatonin levels were 77,1 ± 12,9 at night and 27,2 ± 6,1 in the morning. There was significant change in the diurnal rhythm of the melatonin in the sjögren patient group. Melatonin secretion at night was found to be lower, in the SSc patient group than the healthy control group, but in the morning was found to be higher. Those were statically significant (p<0.001, p<0.001 respectively). In the patient group, there was no statically significant difference between the serum levels in the morning and night. CONCLUSION: In this study, we found that diurnal rhythm of the melatonin changed in sjögren patients. These findings can be due to the effects of melatonin on the inflammatory responses.
Immune exhaustion is associated with development of cardiopulmonary disease in a non-human primate model of HIV-infection (HUM8P.340)
Authors: Kling Heather , Shipley Timothy , Tarantelli Rebecca , Champion Hunter , Morris Alison , Gladwin Mark , Norris Karen
Published: January 2023

Cardiopulmonary disease remains a leading cause of morbidity and mortality in HIV infection, despite widespread use of anti-retroviral therapies. Chronic inflammation due to ongoing viral replication may lead to immune exhaustion, a potential mechanism for the development of chronic obstructive pulmonary disease (COPD) and/or pulmonary arterial hypertension (PAH). Previously, we have shown that simian immunodeficiency virus (SIV) infection of macaques results in PAH and COPD. In the current study, we examined whether lymphocyte exhaustion and/or activation is associated with cardiopulmonary disease in SIV infection. Macaques were intravenously infected with SIVΔB670 (n=6, PAH study) or SHIV89.6P (n=11, COPD study). Blood and bronchoalveolar lavage (BAL) fluid cells from S(H)IV-infected animals were analyzed by flow cytometry. Serial pulmonary function testing and right heart catheterization were performed to assess pulmonary obstruction and mean pulmonary arterial pressure (mPAP), respectively. Increased effector memory CD8+ T (Tem) cells (CD95+CD28-) (p=0.008) and expression of programmed death-1 (PD-1), a marker of exhaustion (p<0.001), in the lung correlated with increased mPAP in SIV-infected macaques. Increased peripheral blood CD8+ Tem cells correlated with worse pulmonary function (p<0.001) in SHIV-infected macaques. These results support the hypothesis that HIV-related cardiopulmonary disease is associated with chronic immune activation and exhaustion.
Anti-HIV, anti-inflammatory and safety profile of the PDB microbicide candidate (HUM8P.342)
Authors: Mueller Yvonne , Fraietta Joseph , Ratner Deena , Boesteanu Alina , Gupta Phalguni , Katsikis Peter
Published: January 2023

Topical microbicides (vaginal gels) are one mechanism to prevent sexual transmission of human immunodeficiency virus type 1 (HIV-1). We show here that a new class of microbicides, abasic phosphorothioate 2’ deoxyribose backbone (PDB) oligomers, has significant anti-HIV-1 activities in human PBMC cell cultures when either the virus or cells are pre-incubated with the compound. HIV infection is also inhibited in an explant model of human cervical tissue. A second function of PDB other than its anti-HIV activity is its capacity to inhibit HIV-triggered TLR7/9 activation. PDB suppresses the release of HIV-induced pro-inflammatory cytokines/chemokines in cultures of human PBMC and ectocervical tissues. We also demonstrated that PDB is safe and non-toxic in in vitro cell cultures and in an in vivo murine vaginal toxicity model. The effect of PDB is stable as this compound retains activity at pH 4.4 and after transition to a neutral pH, following incubation with biological fluids (seminal plasma, vaginal fluids) and a normal vaginal flora Lactobacillus strain. PDB does not affect the growth of Lactobacillus strains. Phosphorothioate 2’ deoxyribose oligomers are novel microbicide candidates with dual-acting anti-HIV and anti-inflammatory activities and an excellent safety and stability profile.
Long term impairment of CD4 T cell responses after sepsis (HUM8P.327)
Authors: Cabrera-Perez Javier , Condotta Stephanie , James Britnie , Rai Deepa , Kucaba Tamara , Badovinac Vladimir , Griffith Thomas
Published: January 2023

Patients that survive the acute stages of sepsis tend to have severely compromised T cell immunity and increased susceptibility to infection. Interestingly, little is known about the mechanism(s) behind decreased CD4 T cell function after sepsis. Using a cecal-ligation and puncture (CLP) model of sepsis, we utilized p:MHCII tetramers to track the recovery of Ag-specific CD4 T cell populations after septic injury, and to probe responses to infection during or after recovery. In agreement with previously published data, we observed a systemic loss of CD4 T cells early on (48h after CLP), but there was no discernible numerical difference in the CD4 T cell compartment of sham- and CLP-treated animals after 30d. Despite this seemingly complete recovery, we detected changes in the distribution of Ag-specific CD4 T cell precursor pools, with quantitative reductions in some populations persisting up to 30d after surgery. Importantly, incomplete recovery of Ag-specific precursor numbers correlated with impaired primary expansion and decreased Ag sensitivity after infection. Moreover, Vβ clonotype composition analysis demonstrated changes intrinsic to an Ag-specific population’s heterogeneity. Our results reiterate the stochastic nature of CD4 lymphopenia during sepsis, and suggest that recovery is accompanied by deleterious alterations to the Ag-specific CD4 T cell repertoire. These findings may explain why patients are disparately predisposed to opportunistic infections after sepsis.
Investigating immunological pathways and diseases with a comprehensive compendium of human data (HUM8P.347)
Authors: Zaslavsky Elena , Gorenshteyn Dmitriy , Fribourg Miguel , Park Christopher , Kleinstein Steven , Sealfon Stuart , Troyanskaya Olga
Published: January 2023

The exponential growth of high throughput immunological data motivates the need to leverage this global effort to guide new investigation and to contextualize domain-specific results. Improved specificity and accuracy over currently utilized approaches such as ontological analysis would be advantageous. To develop such a resource (ImmuNet), we used immunological pathway-guided Bayesian integration of a comprehensive, heterogeneous compendium of human data to assemble functional relationship networks. The utility of ImmuNet was evaluated in various applications. We studied the cellular gene response signature to virus infection, using ImmuNet to guide confirmatory experiments into functionally relevant responses to different viruses. We also demonstrated that ImmuNet can accurately predict disease associated genes in the vicinity of GWAS loci and to explore processes and pathways underlying immune-mediated diseases. By unlocking immunological information captured within the global biomedical research effort, ImmuNet should be widely beneficial to future studies investigating the mechanisms of the human immune system and immunological disease. An interactive user-friendly web interface for investigators is publicly available at immunet.princeton.edu.
Age-associated inflammation enhances macrophage-pathogen interactions in vitro. (INC7P.400)
Authors: Turner Joanne
Published: January 2023

Increasing age is associated with elevated levels of circulating pro-inflammatory cytokines, termed inflammaging. This inflammatory state can modulate a variety of immune functions and contribute to the increased susceptibility of the elderly to numerous infection diseases. Using Mycobacterium tuberculosis (M.tb) as a model pathogen we evaluated the influence of inflammation on macrophage function in vitro using the aged mouse model. We detected elevated basal levels of inflammatory cytokines in the lungs of naïve old mice, and pulmonary macrophages were shown to be in a pre-activated state(elevated mRNA for IRF-1, IRGM1 and CIITA). Furthermore, lung macrophages from old mice had altered trafficking marked by enhanced phago-lysosome (P-L) fusion upon infection with M.tb in vitro, which was refractory to enhancement by IFN-gamma activation. When old mice were placed on a diet of the non-steroidal anti-inflammatory drug (NSAID) ibuprofen for 2 weeks prior to isolation of lung macrophages, this pre-activated phenotype was reversed in old mice. These data demonstrate that the hyper-inflammatory state of the lung in old mice can influence the early interactions of immune cells with M.tb and that this phenotype can be reversed by NSAIDs. These data suggest that inflammaging in the lung could have a beneficial impact on innate immune function during infection, but the long term consequences of this phenotype in the context of M.tb infection are currently unknown.
The role of microbial pattern recognition mechanisms in bacterial-induced autophagy in the social amoeba Dictyostelium discoideum (INC7P.416)
Authors: Snyder Michelle , Fink Marc , Weichseldorfer Matthew , Stephens Kierra , Zapf Ava , Pflaum Katherine
Published: January 2023

Signaling downstream of innate immune pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), results in activation of various responses, including the induction of autophagy. Recent evidence suggests that autophagy plays a protective role against invading intracellular microbes. We have found that upon incubation with bacteria or with the microbial pattern LPS, autophagosomal maturation is stimulated in the model organism Dictyostelium discoideum, which uses bacteria for nutritional purposes. In addition, the rate of bacterial clearance by D. discoideum cells is increased upon exposure of cells to the autophagy-inducing drug rapamycin. Conversely, lysosomal transport of bacteria is decreased in D. discoideum lacking the autophagy protein, Atg1. Although the D. discoideum genome does not encode for TLRs, it does encode for a number of TIR (toll/interleukin-1 receptor) domain-containing proteins, including TirA. Cells lacking TirA are deficient in responses to Gram-negative bacteria. We have found here that TirA plays a role in bacterial-induced generation of reactive oxygen species, a potential stimulant of autophagy. We are investigating the role of TirA as well as the role of reactive oxygen species in pathogen-induced autophagy. These studies are aimed at linking microbial pattern recognition and the induction of autophagy in this model organism to provide insight into conserved mechanisms underlying bacterial detection.
Genetics of differential susceptibility to pneumonic Burkholderia pseudomallei infection (INC7P.424)
Authors: Emery Felicia , Parvathareddy Jyothi , Pandey Ashutosh , Cui Yan , Williams Robert , Miller Mark
Published: January 2023

Burkholderia pseudomallei (Bp) is a pathogenic gram-negative bacterium that causes the severe human disease melioidosis. Bp is listed as a CDC Tier 1 select agent and is considered a potential bioterrorism agent that poses a threat to national security if intentionally released into the human population. Our goal is to use a murine model of Bp infection to gain a better understanding of the interactions between this bacterium and its hosts. We have utilized fully genotyped recombinant inbred BXD mice and a powerful array of complementary computer-based modeling algorithms and databases collectively known as the GeneNetwork. Preliminary studies revealed that Bp infection elicits phenotypically distinct innate immune responses in terms of immune cell recruitment to the lungs, survival and weight loss following pneumonic infection in parental and BXD mice. Preliminary interval mapping of our survival and weight loss phenotypic data using GeneNetwork revealed that survival is a complex trait involving loci on chromosomes 5, 7, and 9 and weight retention involves loci on chromosome 12. Furthermore, we have identified several potential candidate genes within the significant and/or suggestive interval on these chromosomes that appear to correlate with differential susceptibility to Bp infection. These results form the foundation for future work that will significantly increase our understanding of the interactions between Bp and its genetically diverse hosts.
Towards delineating the role of dendritic cells and macrophages in SIV disease control and progression (INC9P.447)
Authors: Swan Zachary , Smith Amanda , Bouwer Anthea , Barratt-Boyes Simon
Published: January 2023

In HIV infection, sustained inflammation and immune activation are signature pathologies of individuals that progress to AIDS. Dendritic cells (DC) and macrophages mediate host inflammatory and antiviral responses and some evidence suggests these cells could be involved in influencing HIV disease outcome, but to what extent is unclear. Here, we longitudinally followed the kinetics of DC and macrophages in peripheral and mucosal tissues from Mamu-B*008+ rhesus macaques intrarectally infected with pathogenic SIVmac251. Mamu-B*008 is a rhesus macaque MHC class I allele highly correlated with control of SIV replication, as greater than 50% of SIV-infected Mamu-B*008+ macaques control chronic phase viral load and do not advance to disease; hence this serves as a model for controlled and progressive HIV disease in humans. We used flow cytometry to access the impact of SIV infection on DC and macrophage proportions, proliferation, and homing to lymph nodes and mucosa. Our preliminary findings reveal a divergent effect of SIV infection on each of the aforementioned DC and macrophage parameters as a function of viral load and tissue compartment. Ongoing experiments will examine the impact of SIV infection on the antigen-presenting cell properties and inflammatory nature of DC and macrophages in the context of SIV disease-progressing and -controlling hosts. These studies should provide clarity on whether DC and macrophages serve a beneficial or detrimental capacity in SIV pathogenesis.
HSV-2 increases TLR4-dependent phosphorylated IRFs and IFN-beta induction in cervical epithelial cells (INM9P.460)
Authors: Li Hui , Liu Hongya , Chen Kai , Feng Wenqiang , Guo Juanjuan
Published: January 2023

The innate immune pathway in response to HSV-2 in its natural host cells remains unclear. Our study has established an in vitro HSV-2 acute infection model with Human Cervical Epithelial (HCE) cells to investigate the role of TLRs-mediated innate immune response to HSV-2. Our previous studies demonstrated that HSV-2 infection up-regulates TLR4 expression and induces NF-kB activity, thereby facilitating innate immune response in human cervical epithelial cells. This process requires involvement of TLR4 adaptors, Mal and MyD88. In the current study, we found that HSV-2 infection increases levels of phosphoryalted IRF3 and IRF7, then regulating expression of type I IFN. As expected, these changes induced by HSV-2 infection depended upon TLR4. To study involvement of the downstream adaptors, we found that overexpression of TRIF and /or TRAM further increased induction of NF-kB activity, while siRNA experiments demonstrated that adaptor molecules TRIF and TRAM were also required for the TLR4-mediated both NF-kB activity and expression of IFN-beta. Our results demonstrate for the first time that IRF3 and IRF7 are both involved in inducing TLR4-dependent IFN- beta expression in response to HSV-2 in its primary infected genital epithelial cells. Thus, TLR4-Mal/MyD88 and TLR4-TRIF/TRAM signaling may synergize and /or cooperate in innate immune response of cervical epithelial cells to HSV-2 infection. (This work was supported by National Natural Science Foundation of China 81072419)
Impaired TLR8 expression toTLR7/8 agonists in patients with chronic hepatitis C virus infection (INM9P.461)
Authors: Lin Tsun-Mei , Lin Chih-Wen , Wang Chiou-Huey , Eng Hock-Liew
Published: January 2023

Toll-like receptors (TLRs) play pivotal roles in the innate immune system and control inflammatory responses and adaptive immunity. TLR7 and TLR8, located in X chromosome, recognize single-stranded RNA and play a significant role in anti-HCV immune response. We consecutively enrolled 50 chronic hepatitis C patients and 50 anti-HCV-negative healthy individuals as controls. The expressions of TLR7, TLR8 and cytokines in peripheral blood before and after TLR7/8 agonist stimulation were measured by RT-PCR and ELISA. Over expression of TLR7 and TLR8 was detected in chronic hepatitis C patients as compared to controls (p < 0.01). Serum levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were also increased in patients (p<0.05). On stimulating the whole blood with R837 (20 μM) and CL075 (20 μM) for 24 hrs, TLR7 and TLR8 expressions were enhanced in healthy controls, but with lower response of TLR8 induction in chronic hepatitis C patients (p<0.01). Increased cytokines production was observed after challenge with TLR7/8 agnonists but no significant difference between the two groups. In conclusion, up-regulation expression of TLR7 and TLR8 and increased proinflammatory cytokines occur in patients with chronic HCV infection, whereas upon agonist stimulation, the patients were demonstrated to modulate immune responses by impairment TLR8 synthesis. The molecular mechanisms of HCV infection and TLR8 hyporesponsiveness need further investigation.