bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin D2 ameliorates acute lung inflammation by activating Nrf2 independent of 15-deoxy-Δ12,14-PGJ2. (P5040)
Authors: Kim Kyun Ha , Han Wei , Sadikot Ruxana , Christman John , Breyer Richard , Chan Jefferson , Urade Yosihiro , Oh Yu-Kyoung , Blackwell Timothy , Joo Myungsoo
Published: January 2023

Since anti-inflammatory activity of prostaglandin D2 (PGD2) is attributed to 15-deoxy-Δ12,14-PGJ2, a byproduct of PGD2, we explored the hypothesis that PGD2, independent of 15-deoxy-Δ12,14-PGJ2, activates NF-E2-related factor 2 (Nrf2), an anti-inflammatory transcription factor, contributing to suppression of lung inflammation in acute lung injury (ALI). Intratracheal delivery of PGD2 decreased neutrophilic infiltration, MPO activity, and the production of pro-inflammatory cytokines in LPS-treated lungs. In contrast, lipocalin-prostaglandin D synthase (L-PGDS) KO mice that produce less PGD2 showed prolonged lung inflammation, highlighting the importance of L-PGDS and PGD2 in suppressing lung inflammation. Two log10 less of PGD2 than 15-deoxy-Δ12,14-PGJ2 was sufficient for activating Nrf2 in RAW 264.7. Similarly, PGD2 was more potent than 15-deoxy-Δ12,14-PGJ2 in expressing Nrf2 dependent genes in mouse lungs. While overexpression of DP1, a receptor of PGD2, enhanced PGD2-mediated Nrf2 activation, siRNA silencing DP1 expression reduced its activation, suggesting that PGD2 uses DP1 in activating Nrf2. Furthermore, PGD2 synergistically suppressed neutrophil infiltration when nrf2 was functionally complemented to the lung of the nrf2-/- mice. Our results show that PGD2 activated Nrf2 independent of 15-deoxy-Δ12,14-PGJ2 and involved DP1. We suggest that PGD2 has a potent anti-inflammatory activity, which is at least in part mediated by activating Nrf2.
Influence of ovariectomy on the cytokine release in acute lung injury induced by LPS in mice (P5052)
Authors: Gimenes-Junior Joao , Vitoretti Luana , Accetturi Beatriz , Lino-dos-Santos-Franco Adriana , Ligeiro-de-Oliveira Ana , Peron Jean , Aloia Thiago , Palermo-Neto Joao , Oliveira-Filho Ricardo , Tavares-de-Lima Wothan
Published: January 2023

Acute respiratory distress syndrome is a multifactorial disease characterized by acute lung injury, hypoxemia and polymorphonuclear cells infiltration. Clinical and experimental evidences indicate that female sex hormones may greatly influence lung inflammation. Previous data from our group revealed that ovariectomy (OVx) blunted LPS-induced lung inflammation in mice after 24 h. Here, we expanded the study and assessed the influence of OVx on the release of cytokines by lung cells after LPS. 7 day-OVx female C57BL/6 mice were subjected to intranasal instillation of LPS or saline. Non-manipulated animals were subjected to the same procedures and served as controls (Sham-OVx). 24 h thereafter, animals were euthanized and lungs removed. The levels of IL-1β, IL-17, IFN-γ and IL-10 were measured. Ovaries removal per se did not alter the cytokines levels in the lung compared with the control group. Moreover, intranasal instillation of LPS increased the cytokines released in the lungs from both OVx and Sham-OVx groups compared to controls. On the other hand, LPS challenged animals had more IL-1β, IL-17 and IFN-γ within the OVx group. This was associated with significantly higher levels of IL-10 in the same group. Lack of female sex hormones led to an increased release of pro-inflammatory cytokines by the lungs, demonstrating its role in modulating the development of acute lung injury induced by LPS in mice.
Role of GM-CSF and IRF5 in steroid refractory asthma (P6247)
Authors: Gordon Rachael , Raundhal Mahesh , Wenzel Sally , Ray Anuradha , Ray Prabir
Published: January 2023

Asthma is a heterogeneous, inflammatory disease of the airways characterized by reversible airflow obstruction and bronchoconstriction. 10% of asthma patients are categorized as severe asthmatics (SA), showing mixed neutrophilic/eosinophilic infiltrate, which is poorly responsive to corticosteroids (CS). Elevated levels of inflammatory cytokines, including GM-CSF, IL-6 and IL-17, are found in the airways of SA. Recently, GM-CSF alone or in combination with LPS, was shown to augment expression of IRF5 in macrophages (Mϕ), promoting Th17-driving cytokines. These observations led us to hypothesize that GM-CSF plays an important role in mediating CS-refractory IL-6 production from Mϕ. In RAW 264.7 cells, LPS-induced IL-6 production was inhibited 65% by CS. However, when LPS was combined with GM-CSF at 20 or 200 ng/ml, CS poorly suppressed IL-6 production (17 % and 10.5% respectively). IRF5 expression was detected in LPS+GM-CSF-treated RAW cells and its expression was augmented in the lung-draining lymph nodes of mice exposed to the allergen, house dust mite. Interestingly, IRF5 expression was significantly higher in bronchoalveolar lavage cells isolated from human SA as compared to that in cells from milder, CS-responsive asthmatics or from healthy controls. These observations suggest an important role of GM-CSF and IRF5 in mediating CS-refractory IL-6 production in innate immune cells that in turn promote CS-unresponsive Th17 development and neutrophilic airway inflammation.
Natural EBV infection in humans induces an expression profile similar to cytokine storm illnesses that is unique from other acute viral infections (P6326)
Authors: Dunmire Samantha , Odumade Oludare , Porter Jean , Bacheler Emily , Balfour, Jr. Henry , Hogquist Kristin
Published: January 2023

Herpesvirus infections are common in humans, and while generally not lethal, lead to lifelong infection associated with cancer and autoimmune disease. To better understand immunity to persistent viral infection, we performed a prospective study in otherwise healthy college students of naturally acquired infection by the gamma herpesvirus, Epstein Barr virus (EBV). Transcriptome analysis defined a striking and reproducible profile during acute infection regardless of disease severity and included genes related to cell cycle, interferon response, and immune function. Even with the highly sensitive approach taken, however, no lasting gene changes were apparent during latent infection. Comparing the EBV response profile to multiple other acute viral infections revealed similarity only to Dengue virus. The signature shared by EBV and Dengue was also present in inflammatory syndrome patients. Interestingly, while EBV induced a type I interferon response, a subset of interferon induced genes, including MX1 and OAS1, were not upregulated, despite these genes being conventional contributors in a type I interferon response. Furthermore, IL1B was downregulated, together suggesting potential mechanisms of viral antagonism. These data provide an important first description of the response to natural herpesvirus infection in humans during multiple stages of infection.
Neutralizing KC (CXCL1), but not IL-6 ameliorates recurrent HSK in vivo. (P6340)
Authors: Stuart Patrick , Yin Xiao-Tang , Del Rosso Chelsea , Potter Chloe , Potter Chloe , West Devin
Published: January 2023

Herpetic stromal keratitis (HSK) is the leading cause of infectious blindness in developed nations and occurs following infection with herpes simplex virus. The mechanism underlying this disease is immune-mediated, involving neutrophils, macrophages, cytokines, and chemokines. We hypothesized that neutralizing the proinflammatory cytokine IL-6 or the chemokine KC (murine CXCL1) would significantly reduce disease in an animal model of HSK. Latently infected mice were reactivated 6 weeks following infection with UV-B light to stimulate recurrent disease. At that time, we administered mAbs against IL-6, KC and control IgG via intraperitoneal injection. We monitored recurrent infection by daily testing tear film for virus for 7 days following reactivation. Corneal disease was evaluated weekly for five weeks. Data shows that neutralizing KC resulted in lower opacity and neovascularization scores for all time points measured when compared to controls. Surprisingly, unlike what is seen in acute HSK, neutralizing IL-6 did not decrease disease. Futhermore, treatment with anti-KC results in significantly less neutrophilic infiltrate of infected corneas. We conclude that CXCL1 and not IL-6 is important in developing recurrent HSK. Furthermore, these results further demonstrate that recurrent and acute HSK share some but not all aspects of disease, and that one common thread is the crucial role that neutrophils play in this potentially blinding disease.
RIG-I receptor regulation of T cell immunity during virus infection (P4361)
Authors: Suthar Mehul , Ramos Hilario , Brassil Margaret , Netland Jason , Chappell Craig , Blahnik Gabrielle , McMillan Aimee , Diamond Michael , Clark Edward , Bevan Michael , Gale, Jr. Michael
Published: January 2023

West Nile Virus (WNV) is an RNA virus that is the leading cause of mosquito-borne encephalitis in the United States. Recently, we demonstrated that MAVS, the central adaptor molecule of the RIG-I like receptor (RLR) signaling pathway, is essential role in programming innate and adaptive immune defenses to control WNV infection. However, the role of the individual RLRs in directing immunity during virus infection has not been clearly defined. WNV infection of Lgp2-/- mice showed increased mortality which corresponded to enhanced virus replication and reduced antigen-specific CD8+ T cells in the CNS at late times during infection. While LGP2 was determined to be non-essential in innate immune responses to WNV infection, LGP2 was required for controlling antigen-specific CD8+ T cell survival and effector functions during virus infection. Adoptive transfer studies of WT and Lgp2-/- CD8+ T cells into Rag-/- mice demonstrated that LGP2 functions in a cell-intrinsic manner to regulate T cell sensitivity to CD95-mediated cell death. Biochemical studies showed that LGP2 expression in CD8+ T cells is regulated through crosstalk between the T cell receptor signaling and interferon signaling pathways. Mechanisms by which LGP2 regulates extrinsic apoptosis signaling and T cell survival will be discussed. Overall, these studies demonstrate a novel for LGP2 in regulating T cell immunity and reveals a broader role for RLRs in programming adaptive immune responses during virus infection.
Licensed and unlicensed natural killer cells show differential immunoregulatory and effector mechanisms during viral infection. (P4367)
Authors: Sungur Can , Zamora Anthony , Tang-Feldman Yajarayma , Baumgarth Nicole , Pomeroy Claire , Murphy William
Published: January 2023

Natural killer (NK) cells are innate lymphocytes with anti-viral and tumor capabilities that can be divided into subsets based on receptor expression patterns. If an NK cell expresses an inhibitory receptor that can bind to self-MHC I molecules it is considered to be “licensed” and fully functional; however, if the cell cannot bind to self it is “unlicensed”. We have observed differential effector and immunoregulatory functions of licensed versus unlicensed cells during influenza and murine cytomegalovirus (MCMV) infections in mouse models. We predict that licensed NK cells have anti-viral properties as well as immunoregulatory characteristics by killing T cell populations. The unlicensed NK cells may serve more of a helper cell role by aiding in tissue regeneration and enhancing dendritic cell (DC) numbers. After influenza or MCMV infection, there is a significant reduction in antigen-specific CD4 and CD8 T cell populations in the presence of licensed NK cells based on flow cytometry. Depletion of unlicensed NK cells reduces the number of DCs in the target tissues and lymph nodes. Additionally, the unlicensed NK cells are the primary producers of IL-22 based on intracellular staining in the damaged tissues after viral infection, aiding in tissue regeneration. Collectively, these data suggest a suppressive role for licensed NK cells to reduce the number of T cells, while the unlicensed serve as helper cells to aid in tissue regeneration and DC expansion during infection.
How MAIT cells control the growth of intracellular mycobacteria (P4381)
Authors: Sakala Isaac , Yankelevich Wei-Jen , Kjer-Nielsen Lars , Fairlie David , Rossjohn Jamie , McCluskey James , Hoft Daniel , Hansen Ted
Published: January 2023

Mucosal associated invariant T cells or MAIT cells have a semi-invariant TCR Vα chain and limited Vβ chains. In addition MAIT cell development is dependent upon the commensal microbiota and the expression of MR1, a highly conserved class Ib molecule of mammals. Mouse and human MAIT cells are activated in an MR1-restricted manner by cells infected with diverse strains for bacteria suggesting a widely shared antigen. Remarkably, human MR1 was recently shown to bind select bacterial vitamin B metabolites capable of activating MAIT cells in an MR1-restricted manner. This finding raised the interesting question of the importance of MR1 presentation of vitamin B metabolites for controlling bacterial infection. To address this question we have developed a mouse model whereby mycobacteria infected macrophages are co-cultured with purified MAIT cells, and control of intracellular bacterial growth is monitored. Using this assay, MAIT cell control of mycobacterial growth was found to depend upon IL-12 secretion by infected macrophages and IFN-γ secretion by MAIT cells. Interestingly, in the absence of infection, MAIT cells secreted IFN-γ in response to recombinant IL-12 alone or antigenic vitamin B metabolites. These findings demonstrate that MAIT cells can be activated by either TCR or non-TCR interactions. The contribution of these two pathways of MAIT cell activation in the context of mycobacterial infection is currently being investigated.
PGI2 inhibits IL-33-induced innate type 2 cytokine responses by natural helper cells (P4343)
Authors: Zhou Weisong , Toki Shinji , Goleniewska Kasia , FitzGerald Garret , Peebles R.
Published: January 2023

Natural helper (NH) cells are newly identified innate lymphoid cells that produce large amounts of type 2 cytokines including IL-5 and IL-13 in response to IL-33. NH cells are critical in protective type 2 immunity against helminth infection and mediate influenza virus-induced airway hypersensitivity. However, how type 2 immune responses of NH cells are regulated is not known. In this study, we tested the hypothesis that prostaglandin I 2, a lipid product formed in cyclooxygenase (COX) pathway in arachidonic acid metabolism, suppresses IL-33-induced type 2 cytokine responses by NH cells. Mouse NH cells (Lin -CD25 +CD127 + cells) were purified by flow cytometric cell sorting from bone marrow and stimulated with IL-33 in the presence of the PGI 2 analog cicaprost or vehicle. We found that cicaprost dose-dependently decreased IL-13 production by NH cells. In addition, when mice deficient of PGI 2 receptor (IP KO mice) were administered with IL-33 to the lung, they had greater IL-5 and IL-13 responses in the lung at 6 h after IL-33 challenge compared to WT mouse controls. These data indicate that PGI 2 and its receptor signaling inhibit NH cell-mediated innate type 2 immune responses and suggest that use of COX inhibiting drugs may increase the risk of developing allergic diseases by augmenting innate type 2 immunity.
Exogenous vitamin D inhibits human pro-inflammatory cytokine responses to TLR-4L but not TLR-8L innate immune activation (P6331)
Authors: Fitch Natascha , Becker Allan , HayGlass Kent
Published: January 2023

Population health studies demonstrate that insufficient/deficient Vitamin D status is strongly associated with increased upper respiratory tract infections. As low vitamin D status is widespread, it is imperative to understand the roles it plays during an immune response. Here we determine the effects of active vitamin D (1,25(OH)2D3) on peripheral blood mononuclear cells (PBMC) cytokine responses elicited by bacterial or viral ligands and explore mechanisms by which vitamin D regulates innate immune responses. Results: PBMC stimulated with TLR-4L and exogenous 1,25(OH)2D3 exhibited decreased pro-inflammatory cytokine production; whereas cultures stimulated with TLR-8L did not exhibit alterations in cytokine production patterns in the presence of supplemental 1,25(OH)2D3. 1,25(OH)2D3 alone slightly reduced TLR-4 but decreased TLR-8 by >75%. Moreover, while TLR-4 ligand stimulation had no effect on VDR levels, TLR-8L stimulation reduced VDR mRNA by 60% (p<0.005). Conclusions: Exogenous 1,25(OH)2D3 markedly reduces inflammatory cytokine production elicited by TLR-4 but not by TLR-8 or RSV activation. TLR-8L strikingly reduce VDR while 1,25(OH)2D3 reduces viral PRR expression, resulting in a synergistic decrease in the capacity of viral stimulated cells to have responses modulated by exogenous vitamin D. The results suggest a need for closer examination of the benefits of exogenous vitamin D supplementation in bacterial versus viral infection.
PGE2 modulates the ability of IFN-γ to inhibit IL-23 production in activated dendritic cells (P6333)
Authors: Rahbari Kate , Adhikary Sabina , Ganea Doina
Published: January 2023

The proinflammatory cytokine IL-23 emerged recently as a key player in autoimmune diseases. Production of IL-23 by pathogen-activated dendritic cells (DC) is regulated by endogenous mediators such as prostaglandin E2 (PGE2) and interferon-γ (IFN-γ). PGE2 stimulates IL-23 and inhibits IL-12 production. In contrast, IFN-γ inhibits IL-23 and stimulates IL-12. In the presence of both PGE2 and IFN-γ, TLR-stimulated DC produce higher levels of both cytokines. Here we analyze the signaling pathways involved in this complex regulation. IRF1, induced by LPS and at high levels by IFN-γ, acts as a positive transcription factor for IL-12p40, and as a negative regulator for IL-23p19. The potent effect of IFN-γ on IRF1 correlates with the inhibition of IL-23 and stimulation of IL-12 in LPS-activated DC. In contrast, PGE2 abolishes IRF1 expression in LPS-stimulated DC, resulting in the inhibition of IL-12 and stimulation of IL-23 production. When both PGE2 and IFN-γ are present, the IRF1 levels are similar to LPS controls, allowing for both IL-23p19 and IL-12p40 expression. In addition, as previously reported by us, the stimulatory effect of PGE2 on p19 transcription is also mediated through the activation of the positive transcription factors CREB and C/EBP. Our findings suggest that the combined presence of PGE2 and IFN-γ in autoimmune conditions results in sustained production of both IL-12 and IL-23, which contribute to the maintenance of a proinflammatory environment.
Flt3 ligand expands and activates myeloid-derived suppressor cells in a STAT3-independent manner (P1074)
Authors: Rosborough Brian , Liu Quan , Raïch-Regué Dàlia , Matta Benjamin , Mathews Lisa , Thomson Angus , Turnquist Heth
Published: January 2023

Introduction: Fms-like tyrosine kinase 3 ligand (Flt3L) promotes dendritic cell (DC) expansion; however, its influence on myeloid-derived suppressor cells (MDSC), an immature population of myeloid cells that suppresses T cell immunity, has not been studied. Methods: BALB/c mice were treated with Flt3L (10 μg/d i.p., 10 d) in the presence or absence of STAT3 inhibitor S31-201 (5 mg/kg/d). Splenic DC and MDSC were isolated by CD11c+ and Gr1+ magnetic bead selection, respectively. Stimulation and suppression of CD3+ T cell responses were assayed by MLR. Syngeneic MDSC were adoptively transferred (5x106) to BALB/c recipients 1 d before C57BL/6 heart transplantation and survival monitored. Results and Conclusions: Flt3L increased MDSC (CD11b+Gr1+) frequency and absolute numbers. MDSC from Flt3L-treated mice potently suppressed CD3+ T cell proliferation greater than control MDSC from naïve splenocytes. Conversely, Flt3L-expanded DC stimulated increased proliferation of allogeneic T cells compared to those from naïve control mice. While STAT3 is considered crucial for MDSC expansion and activation, STAT3 inhibition reduced Flt3L-mediated DC, but not MDSC, expansion. STAT3 inhibition augmented MDSC expansion by Flt3L, without affecting their suppressive capacity. Flt3L-expanded MDSC, but not control MDSC, demonstrated in vivo suppressive activity by prolonging fully MHC-mismatched heart transplant survival. Together, these data identify a novel immunomodulatory function of Flt3L.
The microbiome and IL-10 are critical factors in regulating the number of marginal zone B cells (P1086)
Authors: Dittel Bonnie , Basu Sreemanti , Ray Avijit
Published: January 2023

Marginal zone (MZ) B cell positioning within the spleen allows them to rapidly respond to blood-borne pathogens. Very little is known regarding how MZ B cell homeostasis is controlled. We found a critical role for IL-10 and the microbiome in regulating MZ B cell numbers. Specifically, IL-10-/- mice obtained from Jackson Lab (JAX) but maintained in our colony exhibited a significant increase in the absolute number of MZ B cells. However, mice from JAX had normal numbers of MZ B cells. When we co-housed IL-10-/- JAX mice with our IL-10-/- mice, MZ B cell numbers were significantly increased. These results suggested that the microbiome plays a prominent role in regulating MZ B cell homeostasis. To confirm this, we treated IL-10-/- JAX mice with broad-spectrum antibiotics during the co-housing period and were able to prevent the increase in MZ B cell numbers. In addition, the treatment of our IL-10-/- mice with antibiotics was sufficient to reverse the increase in MZ B cells. One major difference between the two sources of mice is that ours are colonized with helicobacter, a contributing factor to inflammation and colitis. We found that specific colonization of JAX IL-10-/- mice with helicobacter was sufficient to increase MZ B cell numbers. Thus we have uncovered that IL-10 controls a regulatory loop whereby specific bacteria in the gut capable of inducing inflammation drive an increase in MZ B cells that would be beneficial should a breach in the gut result in sepsis.
Interleukin-21-induced granzyme B-expressing B lymphocytes regulate T cells and infiltrate tumors (P1088)
Authors: Lindner Stefanie , Dahlke Karen , Sontheimer Kai , Hagn Magdalena , Kaltenmeier Christof , Barth Thomas , Beyer Thamara , Reister Frank , Fabricius Dorit , Lotfi Ramin , Lunov Oleg , Nienhaus G. Ulrich , Simmet Thomas , Kreienberg Rolf , Möller Peter , Schrezenmeier Hubert , Jahrsdörfer Bernd
Published: January 2023

The role of B cells in tumor infiltrations is controversially discussed. Different studies suggest that certain tumor-infiltrating B cell populations exhibit regulatory potential. Here, we demonstrate that the microenvironment of various solid tumors contains granzyme B (GrB)-expressing B cells adjacent to IL-21-providing T cells. Since GrB-mediated effector T cell modulation is known from regulatory T cells (Treg) and plasmacytoid dendritic cells, we hypothesized the existence of similar mechanisms in B cells. Here we show that IL-21 induces B cells expressing high levels of GrB and controlling T cell proliferation by GrB-dependent degradation of the T cell receptor ζ-chain. Detailed characterization of IL-21-induced GrB+ B cells reveals a CD19+CD38+CD1d+IgM+CD147+ phenotype and expression of additional regulatory molecules including IL-10, CD25 and IDO. Of note, IL-21-mediated GrB induction integrates both BCR- and TLR-mediated signals and is enhanced in the presence of B cell CD5 expression. This is the first report demonstrating that IL-21 induces GrB+ human regulatory B cells, which can be detected in tumor infiltrations, and which may contribute to the modulation of cellular adaptive immune responses by Treg-like mechanisms. Our findings may stimulate the development of novel diagnostic and cell therapeutic approaches to the management of malignant, autoimmune and graft-versus-host pathologies.
Effects of LPS on NF-κB p65 and anti-inflammatory cytokines mRNA expression in MSC (P1096)
Authors: Gang Wang , Haiyan Wang , Hong Liang
Published: January 2023

Abstract Objective To investigation the effects of Inflammatory simulated by LPS on mRNA expression of NF-κB p65, anti-inflammatory cytokines (IL-10, TGF-β3) and IDO in rat bone-marrow MSC. Methods The MSC cultured in vitro was divided into 6 groups: The normal group, the PDTC(100μmol/L) treatment group, the PDTC(100μmol/L)plus LPS(5µg/mL) treatment group and the 3 LPS dose (1, 5, 10μg/mL) treatment groups. After LPS supplement for 4 hr, the total RNA of the MSC was extracted and the mRNA expression of p65, IL-10, TGF-β3 and IDO were detected by RT-PCR. The gray scale was used to compare the mean levels (X±S) of PCR product within groups were determined. Results Compared with the control group, the mRNA expression of NF-κB Subunit p65 and anti-inflammatory cytokines IL-10, TGF-β3 were significant increased in the LPS treated groups (P <0.05). The correlation coefficient between p65 and IL-10, TGF-β3 were 0.844(P<0.05), 0.837(P<0.05) respectively. The correlation coefficient of p65 with IDO was -0.942 (P<0.05). Conclusion 1 LPS stimulation could induce MSC produce more anti-inflammatory cytokines through the activation of NF-κB. 2 IDO was constitutively expressed in normal cultured MSC, and its expression may be down regulated by inflammatory stimulation. This result indicates that there are different immune-modulation pathways in MSC under different microenvironments. Key words MSC; NF-κB; IL-10; TGF β3; IDO
Complement protein C1q influences macrophage-mediated T cell activation (P1062)
Authors: Clarke Elizabeth , Weist Brian , Walsh Craig , Tenner Andrea
Published: January 2023

Complement protein C1q opsonizes apoptotic cells (AC) to enhance ingestion by human monocyte-derived macrophages (Mϕ) before membrane integrity of the dying cell is lost, thus avoiding the release of inflammatory DAMPS and/or autoantigens. Additionally, Mϕ ingesting C1q-bound autologous AC (C1q-polarized Mϕ) produce enhanced anti-inflammatory and reduced pro-inflammatory cytokines. To understand the functional consequences of C1q interaction with Mϕ, we evaluated the induction of PD-L1 and PD-L2, molecules that bind T cell PD-1 resulting in inhibition of T cell activation, and the effect of C1q-polarized Mϕ on the T cell response to antigen stimulation. First, C1q-polarized Mϕ significantly upregulated both PD-L1 and PD-L2. Second, co-culture of C1q-polarized Mϕ with allogeneic T cells in a mixed-leukocyte reaction (MLR) for 3, 5 or 6 days did not modulate overall T cell proliferation relative to Mϕ ingesting AC alone, but decreased CD4+ T cell proliferation at day 3 (n=3) and increased Foxp3+ T cell proliferation at day 5 (n=4) relative to Mϕ ingesting AC alone. These data suggest that C1q-polarized Mϕ can influence T cell activation. C1q deficiency results in systemic lupus erythematosus (SLE) with >90% penetrance, but the molecular mechanisms involved have not yet been determined. Further investigation of C1q-mediated effects on T cell proliferation could lead to the identification of novel target pathways for therapeutic intervention in SLE and other autoimmune diseases.
Regulated expression of anti-apoptotic MCL-1 and A1 controls the population structure of myeloid derived suppressor cells (P1070)
Authors: Haverkamp Jessica , Bronte Vincenzo , Herold Marco , Opferman Joseph , Murray Peter
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) are an immunosuppressive population of immature monocytic and granulocytic cells. MDSCs emerge from the bone marrow as inactive precursors and upon migration to sites of inflammation become activated regulatory cells. However, both the fate and key immunosuppressive subsets within the MDSC pool at inflammatory sites remain unclear. Here we show MDSCs express high amounts of anti-apoptotic MCL-1 and A1. We reasoned if MCL-1 was ablated in MDSC populations, MDSCs would be less suppressive and more prone to apoptotic death. However, MCL-1 deficient MDSCs were more suppressive than wild type (WT) MDSCs because MCL-1 deficient MDSCs were predominately monocytic in lineage. In the absence of MCL-1, granulocytic MDSCs are apoptotic and are not functional suppressor cells. These data define monocytic MDSCs as the key immunosuppressive population. When we sorted purified monocytic MDSCs from MCL-1 deficient mice we found they had reduced suppressive activity and higher apoptosis than sorted WT monocytic MDSCs. Further investigation demonstrated in the absence of MCL-1, regulated A1 expression in monocytic MDSCs is necessary for their full suppressive capacity and survival. Furthermore, we show GM-CSF is a key player in the survival of monocytic MDSCs via A1 regulation. These data have implications for the engineering of MDSCs as suppressors for autoimmune diseases and therapies to selectively ablate monocytic MDSCs in the treatment of cancer.
Altered host immune responses, not lowered growth, are the mechanism of attenuation of Francisella tularensis clpB (P3126)
Authors: Barrigan Lydia , Tuladhar Shraddha , Brunton Jason , Woolard Matthew , Chen Ching-ju , Saini Divey , Frothingham Richard , Sempowski Gregory , Kawula Thomas , Frelinger Jeffrey
Published: January 2023

Bacterial infections typically initiate both innate and adaptive immune responses. Bacterial attenuation is thought of as altered bacterial growth in the presence of constant immune pressure. In vivo screens have identified F. tularensis genes necessary for virulence. While many of these mutations render F. tularensis incapable of intracellular growth, some mutations have no impact on intracellular growth. We hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS mutant clpB (FTL_0094). Our LVS clpB strain is attenuated in pneumonic tularemia yet we did not observe an intracellular growth defect in bone marrow derived macrophages. We showed that LVS clpB induced an altered innate, but equivalent adaptive, immune response compared to LVS. In B6 mice, LVS clpB induced pro-inflammatory cytokine production in the lung early after infection in contrast to wild-type LVS infection. LVS clpB provoked a robust protective adaptive immune response similar in magnitude to LVS, but with increased IFN-γ and IL-17A production in the lung as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of non-essential gene(s) that cause attenuation, like F. tularensis clpB, can help elucidate bacterial immune evasion mechanisms.
Persistence of antibodies targeting B cell epitopes of Plasmodium falciparum VAR2CSA (P3114)
Authors: Babakhanyan Anna , Leke Rose , Fowkes Freya , Taylor Diane
Published: January 2023

P. falciparum infection poses a major health risk for 85 million women during pregnancy, due to placental pathology resulting from the binding of malaria-infected erythrocytes to trophoblasts via the pregnancy-specific parasite adhesin VAR2CSA. We have demonstrated a correlation between high avidity antibodies (Ab) to VAR2CSA and protection from placental malaria. Ab to most malarial adhesins appear to be short lived, requiring exposure to antigen to sustain serum IgG. In the current study, we used serum collected longitudinally from pregnant Cameroonian women receiving antimalarial prophylaxis to help establish the half-life of Ab to VAR2CSA. VAR2CSA IgG levels were determined using a bead-based multianalyte platform. The half-life of IgG to VAR2CSA (5yr) was short compared to that found in pregnant Thai women (142 yr) and for viruses (ex. measles: 457 yr), but longer than for other non-pregnancy associated malarial antigens (ex. MSP-1: 2yr). Differences in IgG half-life between the two sites could be due to differences in malaria transmission: seasonal in Thailand, but year around in Africa. In Africa, frequent exposure to VAR2CSA during pregnancy may cause prolonged antigenic stimulation leading to continuous B cell activation with formation of short-lived plasmablasts and not long-lived B cells. Knowing how long IgG to VAR2CSA persists is important for establishing continuous protective immunity against malaria in pregnant women.
Mechanisms of inhibition of CD8+ T cell immunity during helminth infection (P3051)
Authors: Marple Andrew , Wu Wenhui , Grotenbreg Gijsbert , Gause William , Yap George
Published: January 2023

Upon infection, the protozoan parasite Toxoplasma gondii induces a strongly polarized Th1 cytokine response, necessary for effector differentiation of CD8 lymphocytes. Pre-infection with an intestinal nematode, such as Heligmosomoides polygyrus has been shown to attenuate immune responses to intracellular pathogens and inhibit the development of Th1 responses. Previous studies have shown that mice co-infected with H. polygyrus and T. gondii exhibit suppressed CD8+ T cell responses against T. gondii, yet the precise mechanism by which co-infection impinges upon the priming and differentiation of antigen-specific T cells remains to be determined. Using a novel Kb-peptide tetramer-binding assay for tgd057-reactive CD8+ T cells, we show that co-infected mice develop a diminished antigen-specific response to T. gondii. Co-infected mice developed fewer antigen-specific CD8+ T cells, a decreased fraction of terminally differentiated effector cells, and reduced effector IFN-γ production. Terminally differentiated antigen-specific effector T cells (CD62Llo, KLRG1+) from co-infected mice retained an intrinsic defect in cytokine production, which is associated with the expression of Spry2, an inhibitor of receptor tyrosine kinase signaling. While tgd057-specific CD8 differentiation and effector function remained inhibited in co-infected STAT6-/- and IL-10-/- mice, helminth-induced inhibition of differentiation and effector function was abrogated in co-infected IL-4/IL-10-/- mice.
Programmed death ligand 2 is a regulator of proinflammatory Th17 responses and immunopathology in murine infection with Schistosoma mansoni (P3085)
Authors: Salantes Danielle , Larkin Bridget , Ponichtera Holly , Sharpe Arlene , Stadecker Miguel
Published: January 2023

Schistosomiasis is a CD4 T cell-mediated inflammatory disease caused by parasitic helminths of the genus schistosoma. The main cause of morbidity and mortality in both humans and an experimental mouse model are tissue-lodged parasite eggs. For reasons not yet fully understood, the severity of immunopathology varies widely amongst mouse strains: CBA/J develop severe disease with an elevated Th17 response whereas C57BL/6 develop mild pathology with a Th2-polarized response. In view of several studies showing that Programmed Death-1 and its ligands PD-L1 and PD-L2 suppress a variety of T cell-mediated immune responses, we hypothesized that this co-inhibitory pathway downregulates the granulomatous response to schistosome eggs, thus preventing severe pathology. We now show that infection in BL/6 mice results in a larger population of PD-L2+ CD11c(hi) splenocytes than in CBA mice. Moreover, PD-L2 deficient BL/6 mice display markedly exacerbated hepatic granulomatous inflammation together with a significant increase in Th17 cells, whereas in PD-L1 deficient BL/6 mice these parameters are not significantly different from WT controls. These results suggest that the induction of PD-L2 expression represents a powerful means to control excessive T cell-mediated immunopathology in this major parasitic disease.
A role for B and T lymphocyte attenuator in regulating T cell responses during Tuberculosis (P3088)
Authors: Grover Ajay , Troudt Jolynn , Foster Chad , May Nick , Izzo Angelo
Published: January 2023

B and T lymphocyte attenuator (BTLA) (CD272) is a coinhibitory receptor expressed by most leukocytes, with the highest expression levels observed on T and B cells, and is involved in the maintenance of peripheral tolerance by dampening the activation of lymphocytes. We wanted to determine if BTLA signaling played a role in T cell meditated regulation during tuberculosis infection. The expression of BTLA was examined on activated (CD44hiCD62Llo) CD4+ T cells in lungs, spleen and lymph nodes in mice during pulmonary Mycobacterium tuberculosis infection. The expression of BTLA on activated CD4+ T cells increased at day 10 post-infection in lungs, when compared to naïve mice, but then declined at day 21 post-infection. In the spleen and lymph nodes while the number of cells expressing BTLA increased, the expression of BTLA on these cells did not increase. Although the number of CD4+BTLA+ T cells in the lungs had increased at day 21, these cells expressed lower levels of BTLA than at day 10, suggesting that BTLA expression was down-regulated during this time. We also found an increase in the percentage of activated and effector memory T cells in the organs when BTLA was blocked using an anti-BTLA antibody during early stage of tuberculosis infection. Further, increased bacterial load was observed in the lungs of mice treated with anti-BTLA antibody indicating that BTLA regulates T cell-driven exacerbation of infection during the early stages of tuberculosis.
Perforin expression is required for fatal blood-brain barrier disruption in the Plasmodium berghei ANKA model of cerebral malaria (P3061)
Authors: Johnson Aaron , Johnson Holly , Huggins Matthew , N'Songo Aurelie , Hanson Lisa , LaFrance Stephanie , Jin Fang , Pirko Istvan , Butler Noah , Harty John
Published: January 2023

The most severe clinical complication of Plasmodium falciparum infection is cerebral malaria (CM). Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice is an established model of CM. Previous work has implied a role for CD8 T cells and perforin expression in fatal stages of PbA infection. We therefore infected C57BL/6 and C57BL/6 Perforin-/- mice with PbA and assessed the CNS for cerebral endothelial cell blood-brain barrier (BBB) disruption using FITC albumin leakage into tissue, confocal microscopy and 3D volumetric T2 and T1 gadolinium enhanced MRI. PbA infected C57BL/6 mice: 1.) had extensive disruption of BBB tight junction proteins Occludin and Claudin 5, 2.) developed severe CNS vascular permeability of FITC albumin and gadolinium leakage, and 3.) became moribund. We also observed increased VEGF cytokine expression coinciding with CNS vascular permeability in these animals. Despite having similar CD8 T cell infiltration into the brain, PbA infected C57BL/6 Perforin-/- mice had intact BBB tight junction, were devoid of CNS vascular permeability, presented with normal brain MRI, and had normal behavioral scores on the rotarod assay. This study is therefore the first to demonstrate the critical importance of perforin expression in promoting BBB tight junction alteration during PbA infection. Therefore, development of therapies to block factors related perforin mediated delivery could have significant benefit to the treatment of CM.
Myeloid cell-specific TRAF3-deficient mice exhibit normal myeloid cell development and homeostasis (P1196)
Authors: Lalani Almin , Moore Carissa , Liu Yan , Gerges Jocelyn , Xie Ping
Published: January 2023

Myeloid cells, including monocytes, macrophages and granulocytes, are crucial players of innate immunity and inflammation. These cells constitutively or inducibly express a number of receptors of the TNF receptor superfamily and Toll-like receptors (TLRs), whose signals are transduced by TRAF molecules. In light of the in vitro evidence that TRAF3 is required for TLRs-induced type I interferon production and for CD40-induced IL-12 production in macrophages, we generated myeloid cell-specific TRAF3-/- (M-TRAF3-/-; TRAF3flox/floxLysM+/Cre) mice to investigate the in vivo functions of TRAF3 in myeloid cells. We examined immune cell populations in the bone marrow, spleen, and peritoneal cavity in M-TRAF3-/- mice at the age of 8-12 weeks. No significant differences in the frequencies or numbers of immune cell populations such as B cells, T cells, granulocytes and macrophages were found between M-TRAF3-/- and littermate control (LMC) mice. TRAF3-/- macrophages also expressed similar levels of MHC class II, CD80 and CD86 as compared to LMC macrophages, suggesting that no endogenous activation of macrophages occurs. Our findings indicate that specific ablation of TRAF3 in myeloid cells neither affected myeloid cell maturation nor changed the homeostasis of macrophages and neutrophils. In contrast, activation of macrophages induced by TLRs was altered in the absence of TRAF3. Our ongoing study will further elucidate the in vivo functions of TRAF3 in innate immunity and inflammation.
Signal control of iNKT development and effector lineage differentiation (P1208)
Authors: Zhong Xiaoping , Wu Jinhong , Wang Shang , Shin Jinwook
Published: January 2023

The type I or invariant NKT iNKT) cells are among the first responders in various infectious and inflammatory settings and bridge innate and adaptive immunity. iNKT cells quickly producing numerous cytokines such as IFNγ, IL-17, IL-4, IL-13, and IL-10 to regulates innate and adaptive immune responses. iNKT cells contribute to the pathogenesis of allergy and asthma, autoimmune diseases, graft-versus-host disease, infections, cancer, and obesity as well as other diseases. While it is well established that the iVα14 T cell receptor and IL-15 receptor and numerous transcription factors are critically involved in iNKT cell development and effector lineage differentiation, the signaling events bridging the receptors and the transcription factors are poorly understood. We have recently demonstrated that diacylglycerol kinases α and ζ play crucial roles in iNKT cell development and that DGKα and ζ negatively control not only the well-known Ras-Erk1/2 and PKCθ-IKK-NFκB pathways but also mTOR signaling in T cells. We now reveal that mTOR signaling and its tight regulation play crucial roles in iNKT cells as deficiency of either mTOR or its regulator TSC1 profoundly impacts iNKT cell development and effector lineage differentiation.
Comparative analysis of different influenza virus strains (H1N1) in the pathogenesis and immune cell infiltration in the lungs in a mouse model. (P6192)
Authors: Kumar Sandeep , Biswas Moanaro , Deventhiran Jagadeeswaran , Elankumaran Subbiah
Published: January 2023

Influenza viruses cause annual seasonal epidemics around the world. Influenza pandemics of varied severity have been reported over time, among which, the 1918 pandemic was most severe. In the present study we sought to explore the pathogenesis and the infiltration of key immune cells in the murine lung following infection with A/PR/08/1934 (mouse adapted seasonal), A/New Jersey/11/1976 (localized epidemics) and A/Ca/04/2009 (pandemic) influenza viruses. We also sought to examine the role of hemmaglutinin (HA) glycoprotein of epidemic and pandemic influenza virus and of a single nucleotide mutation (D222G) in HA gene in altering disease pathology in infected animals. Animals were infected intra-nasally and scored daily for clinical signs (lethargy, ruffled fur and loss in body weight) for fifteen days. Pandemic virus infected mice were first to show clinical signs, though animals recovered soon. Clinical signs were observed after 48hrs post infection in animals infected with mouse adapted seasonal influenza but these animals failed to recover and 100% mortality was observed on day 5-post infection. No clinical signs were observed in animals infected with epidemic influenza virus strain. Lung samples were collected from infected animals on days 1, 3 and 5-post infection and were analyzed for virus titers, immune cells infiltration, pro-inflammatory cytokines and histopathology. Results of these analyses will be discussed.
A novel mechanism regulating anti-viral humoral response: interplay between IL-21, LAPC and TFH in anti-IAV humoral response. (P6174)
Authors: Yoo Jae-Kwang , Fish Eleanor , Braciale Thomas
Published: January 2023

The humoral immune response to most respiratory virus infections plays a prominent role in virus clearance and is essential for resistance to reinfection. T follicular helper (TFH) cells are believed to support the development both of a potent primary antibody response and of the germinal center response critical for memory B cell development. Using a model of primary murine influenza A virus (IAV) infection, we demonstrate that a novel APC, the LAPC (Late-activator APC), promotes the TFH response in the draining LNs (dLNs) of the IAV-infected lungs. LAPCs migrate from the infected lungs to the dLN “late,” i.e., 6 d after infection, which is concomitant with TFH differentiation. LAPC migration is CXCR3-CXCL9-dependent, and LAPC triggering of TFH cell development requires ICOS-ICOSL-dependent signaling. In IAV infection, the host immune system control this LAPC-mediated TFH differentiation via IL-21.We have demonstrated that IL-21, a well known TFH differentiation inducing factor, produced by NK T-cells in the dLN acts on lymph node resident T cells to stimulate TNF-α release by the T-cells. TNF-α in turn stimulates dLN resident dendritic cells to secrete CXCL9, a potent chemotactic stimulus for migration of CXCR3 expressing lung resident LAPC into the dLN. It is then the migrant LAPC which acts to modulate TFH differentiation during respiratory viral infection. Our results reveal a novel and previously unsuspected mechanism for regulating anti-viral TFH and humoral responses.
SLAT-IP3R interaction: implications and functional relevance in T cell activation and calcium signaling (P1172)
Authors: Fos Camille , Becart Stephane , Boehning Darren , Yule David , Altman Amnon
Published: January 2023

SWAP-70-like adaptor of T cells (SLAT) is a guanine nucleotide exchange factor for Rho GTPases that regulates the activation and differentiation of CD4+ T cells by controlling the Ca2+-NFAT signaling pathway. In response to T cell receptor (TCR)/CD28 costimulation, SLAT promotes calcium signaling by regulating calcium release from endoplasmic reticulum (ER) stores. Hence, SLAT-deficient T cells display a severe defect in Ca2+ release from the ER, despite normal activation of PLCg1 and intact production of inositol-1,4,5-trisphosphate (IP3). Here we report a direct and TCR-induced association between SLAT and the (IP3) receptor type 1 (IP3R1), an ER-resident calcium channel, which mediates calcium release from intracellular stores. Using biochemical approaches, we found that both a N-terminal domain, containing an EF-hand motif, and the pleckstrin homology (PH) domain of SLAT bind directly a defined ~20 amino acid region in the regulatory domain of IP3R1. Subcellular fractionation analysis showed that SLAT is present in the ER compartment. Altogether, these data prompt us to hypothesize that SLAT is required for proper IP3R function in T cells, reflecting its direct interaction with the IP3R. Current studies, including strategies aimed at blocking and disrupting the SLAT-IP3R1 interaction, are aimed at determining the biological significance of this novel interaction and its importance for the overall TCR/CD28-induced Ca2+ signaling pathway in T lymphocytes.
Pathogenicity of IFN-γ+ Th17 cells is independent of T-bet (P4128)
Authors: Duhen Rebekka , Glatigny Simon , Arbelaez Carlos , Blair Tiffany , Oukka Mohamed , Bettelli Estelle
Published: January 2023

It is now established that T helper (Th) cell subsets, particularly Th17 cells, are more dynamic than originally anticipated and can acquire the expression of other lineage effector cytokines. However, the molecular cues that dictate their plasticity are unknown. We show that IL-23 plays a central role in the plasticity of Th17 cells and is essential for the generation and propagation of IFN-γ-producing Th17 (IL-17+ IFN-γ+) cells. During the development of experimental autoimmune encephalomyelitis (EAE) IL-17+ IFN-γ+ T cells are preferentially expanded in the central nervous system (CNS). Using an IL-17A-IFN-γ double reporter mouse and I-Ab/MOG38-49 tetramer, we determined that they displayed characteristics of pathogenic T cells, such as GM-CSF expression, and contained the highest number of MOG-specific T cells. Interestingly, the Th1-associated transcription factors T-bet, STAT1 and STAT4 were not involved in the induction of IFN-γ by Th17 cells in vitro. Moreover, in contrast to previous studies showing a role for T-bet in Th1 and Th17-mediated EAE, we demonstrate that T-bet expression in T cells is not required for the emergence of CNS-infiltrating IL-17+ IFN-γ+ T cells and the development of Th17-driven EAE. Our results suggest the existence of different epigenetic programs that regulate IFN-γ expression in Th1 and Th17 cells.