bluuebunny/crossref_metadata_2025 huggingface dataset:
ICOS-expressing Treg are required for resolution of CD8-mediated lung injury (P2198)
Authors: Gardiner Gail , Wu Qiang , Wagner Sarah , Shilling Rebecca
Published: January 2023

Long-term survival of lung allograft recipients can be hindered by the development of obliterative bronchiolitis (OB), a condition that has been linked to acute rejection. CD8+ T cells contribute to acute rejection by inducing lung epithelial injury; however, the mechanisms for mitigating this type of injury are unclear. Using an in vivo model of CD8-mediated lung rejection, we have found bystander CD4+ T cells are required for resolution of acute inflammation. The percentage of CD4+Foxp3+ Treg is significantly higher in inflamed lungs compared to controls, and the majority of these cells are ICOSHi in inflamed lungs. To determine the role ICOS expression plays in the ability of Treg to control inflammation, we investigated Treg cytokine expression levels and found WT Treg express more Il10 than Icos-/- Treg when stimulated in vitro. IL-10 production is also enhanced in WT Treg compared to Icos-/- Treg. Additionally, we saw WT Treg have enhanced proliferation compared to Icos-/- Treg. In vivo, we found Icos-/- CD4+ T cells are not sufficient for resolution of acute inflammation; however, addition of WT Treg rescues this defect. Together, these data suggest ICOS-expressing Treg are required for resolution of CD8-mediated lung injury due to a possible increase in IL-10 production or an enhanced ability to proliferate in vivo. Augmenting ICOS function on Treg after transplantation may protect the lung from acute rejection and prevent OB.
Granulocyte derived cationic peptide LL-37 prime hematopoietic progenitor cells through up-regulating glucose uptake and AMPK signaling (P2202)
Authors: Lee Hakmo , Park Ho Seon , Choi Ok Kyung , Park Jongwoo , Oh Ju Eun , Lee Won-Woo , Chung Sung Soo , Jung Hye Seung , Park Kyong Soo
Published: January 2023

It has been reported that activated granulocytes derived cationic peptide LL-37 positively prime responsiveness of hematopoietic progenitor cells (HPCs) to CXCL12 gradient through an ambiguous pathway. The goal of this study was to elucidate the mechanism of enhanced migration of LL-37 primed HPCs to CXCL12 gradient. We performed in vitro migration assay and found out that LL-37 pretreatment enhanced migration of HPCs to CXCL12 only in the presence of D-glucose, but not in the absence of glucose or in the presence of glucose uptake inhibitor, 2-deoxy-D-glucose (2-DG). This signifies that LL-37 may affect intracellular energy production machinery. So we tested if the priming effect of LL-37 is related to mTOR signaling since mTOR is a central regulator of nutrient sensing and glycolysis promoting glucose uptake and ATP production to match energy demands for cell migration. As we expected, LL-37 induced glucose uptake in mTOR signaling dependent manner. Also, LL-37 induced the activation of nutrient sensing AMPK and its downstream substrates. In line with this, pretreatment of AMPK activator AICAR promoted migration of HSPCs as well. These findings demonstrate that the priming mechanism of LL-37 is closely associated with increasing ATP production and the optimization of glucose uptake and ATP production for HPCs prior to transplantation would be particularly important in HPCs transplantation where the number of cells available for transplantation is usually limited.
Regulatory T cells in prevention of islet-graft rejection in humanized mice (P2230)
Authors: Yadav Mahesh , Szot Greg , Russ Holger , Guo Tingxia , Stephan Stephen , Kroon Evert , Brandon Eugene , D'Amour Kevin , Hebrok Mathias , Bluestone Jeffrey
Published: January 2023

Regulatory T cells (Tregs) are potent suppressor of adverse immune reaction and have shown lot of promise in the preclinical studies for the treatment of autoimmune diseases and organ transplantation. In the current study we tested if ex vivo expanded human Tregs can be used to enhance the engraftment of human islets. To test this we developed a humanized mice model by injecting immune deficient NSG mice with human PBMCs or by injecting fetal-liver derived human CD34+ stem cells. Mice were transplanted with purified human islets or human embryonic stem cells (hESC)-derived islets and followed for graft function by measuring serum levels of human c-peptide. We demonstrate that hESC-derived islet graft or purified human islets were rejected by allogeneic PBMCs, which could be prevented by co-stimulation blockade using CTLA-4Ig and anti-CD40L antibodies. Adoptive transfer of ex vivo expanded human Tregs also rescued the rejection albeit not completely. We are currently generating Ag (islet Ag or allo-Ag)-specific Tregs to test their efficacy in preventing islet graft rejection in a mouse expressing human HLA (DR 0401). Our results indicate that ex vivo expanded Tregs can prevent islet graft rejection by allogeneic PBMCs and that humanized mouse could be an excellent model to test the safety and preclinical efficacy of different Treg population.
Graft acceptance achieved through TIM-4 blockade is dependent on interruption of dendritic cell TIM-4 costimulatory signaling (P2136)
Authors: Yeung Melissa , McGrath Martina , Shimizu Tetsunosuke , Snawder Benjamin , Ueno Takuya , Najafian Nader
Published: January 2023

TIM-4 on DCs and macrophages functions as a costimulatory molecule and PS receptor, mediating phagocytosis of apoptotic cells (AB). The relative importance of these functions in alloimmunity remains unclear. Combined treatment with anti-TIM-4 mAb and CTLA4Ig led to full-mismatch cardiac allograft acceptance (MST>100vs30d,p<0.001), with Treg expansion, decreased Teffector generation, but 10-fold increase in splenic AB. Further characterization of this aTIM-4 Ab showed it impairs AB uptake by macrophages but not DCs. In contrast, interruption of DC TIM-4 led to Treg expansion/induction. Since disruption of AB clearance is pro-inflammatory, we hypothesized that blockade of DC TIM-4 costimulation promotes GS, whereas blockade of its phagocytic function may be detrimental. To test this, we treated recipients with CTLA4Ig +gadolinium (blocks global phagocytosis) +/- aTIM-4. Gadolinium accelerated rejection, but addition of aTIM-4 still led to a 3.5-fold increase in GS (MST 30vs8d,p<0.001). Finally, to confirm that interruption of DC TIM-4 costimulatory function is the predominant mechanism leading to graft acceptance, CD11c-DTR chimeras (for long-term DC depletion) were transplanted. Consistently, we found GS achieved by TIM-4 blockade is attenuated by near-complete DC depletion (MST 26vs20d,ns). In summary, TIM-4 appears to have opposing roles in alloimmunity. Its function as a costimulatory molecule on DCs promotes rejection, whereas its role as a PS receptor may be protective.
Poly(dG) oligo deoxynucleotide conjugated protein vaccine specifically targets skin APCs and induces robust T cell immunity in vivo. (APP3P.110)
Authors: Toussaint Helene , Ciccotelli Jo Erika , Erdos Geza , Carey Cara , Watkins Simon , Larregina Adriana , Falo Louis
Published: January 2023

The efficacy of current vaccines results from the induction of protective antibodies. However we still lack efficacious vaccines against many infectious agents that require durable and protective T cell immunity. Efficient and selective delivery of antigen and adjuvant to APCs in vivo remains a critical challenge to vaccine design. To accomplish both of these goals with a single delivery strategy we have designed an antigen and adjuvant delivery platform based on simple and direct coupling of Poly(dG) oligo deoxynucleotides to protein antigens. Initially we found that immunization of naïve animals by direct injection of conjugates of Poly(dG) and Ovalbumin, Poly(dG)-OVA induces priming of specific CTL responses, differentiation of naïve CD4 T cells into Th1 cells, induction of specific memory T cell responses and generation of specific antibody responses, in a scavenger receptor-dependent manner. Applying this strategy to HIV, we immunized mice with conjugated Poly(dG)-Gag and demonstrated potent induction of Gag-specific CTL and Th1-biased CD4 T cells immune responses in vivo. Further, in human models we show that intradermal injection of Poly(dG)-antigen conjugates result in efficient uptake by human dermal DCs that acquire potent T cell stimulatory functions. Our data suggests that the Poly(dG)-protein conjugate vaccine platform can both selectively target antigens to skin APCs and function as an APC-directed adjuvant to induce broad based cellular and humoral immunity.
Selective CB2 receptor agonist Gp1a attenuates EAE through modulating CD4 T cell differentiation and immune cell infiltration in the CNS (BA8P.131)
Authors: Kong Weimin , Li Hongbo , Tuma Ronald , Ganea Doina
Published: January 2023

CB2 agonists have beneficial effects in experimental autoimmune encephalomyelitis (EAE). Our previous studies showed that the CB2 selective agonist O-1966 inhibited EAE development and leukocyte rolling and adhesion to CNS microvasculature. Here we report that Gp1a, a highly selective CB2 agonist, inhibits EAE development following early treatment and improves recovery following late treatment. Gp1a treatment resulted in lower levels of demyelination, axonal loss, and immune cell infiltration. Our results indicate that early Gp1a treatment inhibits Th1 and Th17 and promotes Th2 and Treg differentiation both in vivo and in vitro. We confirmed that Gp1a inhibited Th1 and Th17 differentiation both in non-polarizing and polarizing conditions. Following later treatment, Gp1a reduced the accumulation of pathogenic immune cells in the CNS, which was associated with reductions in the expression of CNS chemokines and adhesion molecules and of chemokine receptors in T cells. We concluded that at least two mechanisms were involved in the beneficial effects of Gp1a in EAE, an early effect on Th1/Th17 differentiation and a later effect on the accumulation of pathogenic immune cells in the CNS. The combined effect of CB2 agonists on Th17 differentiation and immune cell accumulation into the CNS, suggest the CB2 receptors are potential therapeutic targets in neuroinflammation.
Tissue specific antigen expression shapes T cell repertoire and autoimmune potential (BA8P.133)
Authors: Martinez Ryan , Evavold Brian
Published: January 2023

In the following studies we have identified myelin oligodendrocyte glycoprotein (MOG) as one of the first antigens capable of inducing antigen-specific thymic derived Foxp3+ T regulatory cells (tTregs) and influencing the autoimmune disease potential of the MOG-specific CD4+ T cell polyclonal repertoire. By using MOG38-49:I-Ab tetramer enrichment we have been able to track MOG-specific CD4+ T cells in naive mice through experimental autoimmune encephalomyelitis (EAE). We have found the lack of MOG expression causes a lack of naïve MOG-specific tTreg cell diversion, but no significant difference in the number of MOG-specific effectors. Even though the MOG-specific effectors start at the same numbers in both WT and MOG deficient mice, the MOG-specific CD4+ T cells expand 5-10 fold greater in MOG deficient mice, have a consistently reduced frequency of tTregs and a higher TCR affinity. Upon adoptive transfer into MOG-expressing mice, donor CD4+ T cells from MOG deficient mice cause more rapid onset and increased severity of paralysis, which is correlated with increased expansion and TCR affinity of MOG-specific CD4+ T cells. This data shows the expression of self-antigen during T cell development generates a CD4+ T cell repertoire with an increased frequency of tTregs and a reduced per cell autoimmune potential, demonstrating the importance of tissue specific antigen expression to induce tolerance in T cell responding to self-antigen.
An unexpected role of Smad4 to control autoimmunity and T cell function (BA8P.125)
Authors: Wan Yisong , Gu Aidi , Wang Yunqi
Published: January 2023

Smad-dependent signaling pathway is known to mediate TGF-beta signal in non-lymphoid cells. How this pathway controls T cell function remains poorly understood. In particular, the function of Smad4 (co-Smad) in T cells is perplexing. On the contrary to the prediction that Smad4-deficient mice will pheno-copy TGF-betaR-deficient mice and develop aggressive autoimmunity, Smad4-deficient mice are phenotypically grossly normal. Two explanations may account for such observations. One is that Smad4 is dispensable for TGF-beta function in T cells. The other is that, while Smad4 is important for TGF-beta function in T cells, it is also critical to promote T cell function to offset its TGF-beta-dependent function. To test these possibilities, we generated DKO mice, where both TGF-betaR and Smad4 are deficient in T cells. Much to our surprise, Smad4 deletion rescued the lethal autoimmunity of TGF-betaR-deficient mice. DKO mice phenocopy Smad4-deficient mice. These findings suggest that the second explanation, albeit paradoxical, is correct. Further investigation revealed that, Smad4 is indeed required for TGF-beta function in T cells and, unexpectedly, it is also important to promote T cell function. These findings therefore identify a novel function of Smad4 critical for T cell function and autoimmunity. Here, we will discuss in detail these new findings and the potential underlying mechanisms.
Role of neutrophils in pulmonary vaso-occlusion during sickle cell disease acute chest syndrome (CAM3P.200)
Authors: Bennewitz Margaret , Gladwin Mark , Sundd Prithu
Published: January 2023

Sickle cell disease (SCD) is a hematological disorder that leads to sickling of red blood cells within the microcirculation. The sickle red blood cells (sRBCs) are not only less deformable, but also express α4β1, ICAM-4, Sialyl Lewis X and glycolipids not expressed on non SCD-RBCs. These rigid sRBCs can get sequestered in the microcirculation to cause painful vaso-occlusive crisis (VOC) in SCD patients. In the lung, VOC can contribute to acute chest syndrome, which is a form of acute lung injury. Several factors including the pro-adhesive sRBCs, the large marginated pool of neutrophils, and the hyper-inflammatory state present in SCD can contribute to pulmonary VOC. Although neutrophil-RBC-endothelium interactions are known to play a role in systemic VOC, the cellular mechanism that drives pulmonary VOC remains unknown. Intravital study of the lung microcirculation of live BERK or Townes SCD mice is conducted using two-photon microscopy. Prior to observation, mice are challenged with lipopolysaccharide or PBS to cause lung inflammation and administered (iv) with fluorescent anti-mouse Gr-1 and Ter-119 antibodies to label circulating neutrophils and RBCs, respectively. Vaso-occlusion is defined as cellular aggregation and stasis of blood flow within the lumen of pulmonary capillaries. Preliminary results reveal that circulating macro-aggregates of neutrophils and RBCs may play a role in pulmonary VOC.
The effect of norepinephrine on CD8 T cell subsets in healthy human adults (CCR3P.215)
Authors: Slota Christina , Weng Nan-ping
Published: January 2023

Chronic exposure to stress has detrimental effects on the adaptive immune response. Studies in vitro show that exposure of norepinephrine (NE), a stress hormone, decreases proliferation and functionality of T cells. T cells consist of several different types of cells and each of them serves distinct functions during an immune response. It is unknown how NE reduces functionality of T cells and whether NE has different impact on different types of T cells. Here, we assessed the effect of NE on CD8 T cell function in humans in vitro. We found that not all subsets of CD8 T cells respond the same to NE exposure; memory cells including central memory (Tcm) and effector memory (Tem) were more significantly affected by NE compared to naïve (Tn) CD8 T cells in terms of gene expression of cytokines (IL-2, IFNG, and TNF) important to proliferation and function in the adaptive immune response. Furthermore, expression of NE receptor, Beta-2 adrenergic receptor (B2AR), was higher in freshly isolated memory CD8 T cells (both Tcm and Tem) compared to Tn CD8 T cells. Together these findings show that memory CD8 T cells are more susceptible to NE induced reduction of cytokine production. Currently we are analyzing the effect of NE on general gene expression in Tcm and Tem cells as well as examining histone modifications in these cytokine loci to determine if NE effect via chromatin changes in CD8 T cells.
CCR4-mediated interactions between thymocytes and thymic dendritic cells are required for complete negative selection to self-antigens. (CCR3P.207)
Authors: Hu Zicheng , Jones Jessica , Ehrlich Lauren
Published: January 2023

Clonal deletion of auto-reactive thymocytes is crucial for establishing central tolerance. To mediate negative selection, thymic epithelial cells and dendritic cells (DCs) present self-antigens to thymocytes. Thymocytes that undergo high avidity TCR-mediated interactions with self-peptide:MHC complexes undergo apoptosis to prevent autoimmunity. DCs have been shown to be essential for negative selection. However, the molecular mechanisms governing interactions between thymocytes and DCs are not well characterized. In this study, we find that a chemokine receptor, CCR4, is required for efficient clonal deletion. CCR4 is expressed by post-positive selection thymocytes; CCR4 ligands are expressed by thymic DCs, suggesting CCR4 signaling could mediate interactions between these cell types. Using bone marrow chimeras, we find that CCR4 deficient polyclonal thymocytes fail to undergo efficient clonal deletion, but increase the differentiation to Tregs. While CCR4 deficient OTII TCR transgenic thymocytes also display defects in clonal deletion to endogenous antigens, they undergo efficient deletion to their high affinity ovalbumin antigen. This suggests that CCR4 is not required for TCR-signaling mediating apoptosis, but is required for efficient interactions with thymic DCs that present rare or low affinity antigens. Consistent with defects in clonal deletion, CCR4 deficient mice are prone to autoimmunity, demonstrating that CCR4 is essential for central tolerance.
CD5-CK2 binding domain regulates TCR signals and apoptosis during thymic development. (HEM4P.232)
Authors: Mier Aguilar Carlos Alberto , Raman Chander , Soldevila Gloria
Published: January 2023

CD5 has been previously shown to play an important role in thymic development by downregulating TCR signaling. The receptor contains two major signaling domains in its cytoplasmic tail. The first domain is a functional ITIM domain that recruits proteins such as Ras-Gap, PI3K, Zap 70 and others. The second domain is a CKII Binding Domain (CD5-CKII BD), which in peripheral T cells promotes survival, proliferation and Th differentiation. However, the role of these CD5 signaling domains in early T cell development is poorly understood. In this work we determined the function of CD5-CKII BD in differentiation of thymocyte subpopulations, activation of TCR downstream molecules and regulation of apoptosis using a knock-in mouse in which the CK2 BD is selectively ablated (CD5ΔCKII-BD). Our data show that in non TCR-Tg and in OTII TCR-Tg the loss of CD5-CKII signaling results in a decrease in DP populations in proportion and total cell number and an increase in percentage of CD4+ thymocytes. This reduction in DP thymocytes of OTII.CD5ΔCKII-BD mice was associated with increase in antigen-induced acute activation of ERK phosphorylation relative to OTII.wild-type mice. Additionally, DP and CD4+ thymocytes in CD5ΔCKII-BD mice had increased apoptosis along with elevated levels Caspase 3. Collectively, these results demonstrate that CD5-CKII BD is an important regulator of TCR signals during thymic development and also is a major regulator of the apoptotic events in the thymus.
Peroxisome proliferator-activated receptor-gamma expression in dendritic cells determines immune tolerance versus immune activation (HYP6P.256)
Authors: Khare Anupriya , Chakraborty Krishnendu , Raundhal Mahesh , Morse Christina , Ray Prabir , Ray Anuradha
Published: January 2023

Dendritic cells (DCs) play a central role in regulating immune tolerance and activation but the molecular axis within DCs that dictates immune outcome is unknown. We investigated the role of Peroxisome proliferator-activated receptor-gamma (PPARγ) in DCs in establishment of immune tolerance in the airways using an experimental model. Using mice with selective deletion of PPARγ in CD11c+ cells (PPARγΔ) to ablate PPARγ expression in DCs, we show that DC-specific PPARγ expression exerts a dual role whose collective goal in response to inhaled antigen is to not only promote de novo Foxp3 expression in T cells but to actively dampen T effector cell development. We demonstrate that PPARγ plays a crucial role in enhancing aldh1a2 expression in lung CD103+ DCs under tolerogenic conditions and when tolerized PPARγΔ mice were antigen-challenged, a complete loss of immune tolerance with increased neutrophil-dominated airway inflammation was observed. Absence of PPARγ promoted the expression of multiple pro-inflammatory cytokines such as IL-6 and IL-23 in DCs, limited Foxp3 induction but augmented IL-17 production in T cells with significant increase in the frequency of dual Foxp3+RORγt+ CD4+ T cells in PPARγΔ mice. In summary, our study identifies PPARγ as a central regulator in DC-mediated programming of the Foxp3/RORγt balance in CD4+ T cells dictating tolerance versus inflammation upon antigen provocation.
ILC2 contribute to ozone-induced airway hyperresponsiveness in obese mice (HYP6P.266)
Authors: Mathews Joel , Wurmbrand Allson , Umetsu Dale , Shore Stephanie
Published: January 2023

Many obese asthmatics have non atopic asthma, but the mechanistic basis is poorly understood. Ozone (O3), a common air pollutant, is an asthma trigger that causes airway hyperresponsiveness (AHR) and neutrophilic inflammation. O3-induced AHR and neutrophilia are augmented in obese vs lean mice. Innate lymphoid cells type 2 (ILC2) may be driving these responses. To address this hypothesis, lean WT and obese db/db mice were exposed to room air or O3 (2 ppm) for 3 h. 24 h later, we measured AHR and performed bronchoalveolar lavage (BAL). BAL IL-5, IL-13 and IL-33 were increased by O3 in obese but not lean mice. IL-5 and IL-13 can drive AHR and are secreted by ILC2. IL-33 signaling through ST2 drives activation of ILC2. Therefore, we pretreated db/db mice with ST2 blocking antibody (provided by Dirk Smith, Amgen) prior to O3 exposure. Blocking ST2 decreased O3-induced AHR, baseline lung mechanics, neutrophilia and BAL IL-5 in db/db mice. Anti-IL-13 also decreases BAL neutrophils and lung mechanics in obese mice after O3. IL-23, IL-7, IL-22, and IL-17A were also increased by O3 in obese mice, suggesting the possible involvement of ILC3, a cell activated by IL-7 and IL-23 that secretes IL-17A and IL-22. Blocking IL-17A in a separate cohort also decreased O3 induced AHR and neutrophilia in obese mice. Taken together, our data indicate that obese mice exposed to O3 have increased activation of ILC2 and ILC3 and that these cells are driving the asthma phenotype in these mice.
Limited dependence of IL-4-regulated keratinocyte differentiation and gene expression on STAT6 (HYP6P.261)
Authors: Serezani Ana Paula , Bozdogan Gunseli , Walsh Daniel , Krishnamurthy Purna , Sehra Sarita , Goenka Shreevrat , Spandau Dan , Kaplan Mark
Published: January 2023

Epidermal Differentiation Complex (EDC) genes are critical for barrier function in the skin, and Th2 cytokines that promote allergic inflammation inhibit EDC gene expression in keratinocytes. To further define the effects of the atopic immune response on keratinocyte differentiation, we investigated IL-4-mediated changes in gene expression in human keratinocytes via RNA-seq. Human keratinocytes were differentiated for 2 days with calcium chloride and then stimulated with IL-4 for 24 hours, or keratinocytes were differentiated with calcium chloride and chronically stimulated with IL-4 for 5 days. At day 2, IL-4 increased expression of genes associated with cell cycle, and decreased expression of genes involved with cell adhesion. At day 5 of differentiation, IL-4-stimulated keratinocytes showed increased expression of genes involved with cell projection and adhesion, and decreased expression of genes that participate in wound repair, epidermis development and host defense. STAT6 inhibition by siRNA significantly altered the expression of less than 20% of genes induced by IL-4. Thus, although IL-4 dramatically alters keratinocyte gene expression and differentiation, only a subset of this response is dependent upon STAT6, and eliminating STAT6 expression has IL-4-independent changes in gene expression. This suggests that keratinocyte gene regulation by IL-4 requires additional molecular pathways.
The IL-17-independent role of IL-23 in severe asthma (HYP7P.300)
Authors: Manni Michelle , Mandalapu Sivanarayana , McHugh Kevin , Alcorn John
Published: January 2023

Asthma is a common respiratory disease affecting approximately 300 million people worldwide with no current preventions or cures. Th17 cells are known to be influential in asthma pathogenesis, especially in asthmatics with severe disease that fail to respond to glucocorticoid therapy. Interleukin-23 is critical for the maintenance and full acquisition of Th17 cells, but very little is known about its direct effects outside of IL-17 signaling. This study seeks to investigate novel mechanisms by which IL-23 regulates allergic airway disease. To do this, BALB/c SCID and Rag2-/-γc-/- mice were treated with adenoviral IL-23 or control and airway disease was assessed 3 days later. Preliminary data show that exogenous IL-23 induced airway neutrophilia and pulmonary expression of genes related to asthma pathogenesis and Th17-related cytokines and chemokines, while it increased airway resistance in response to methacholine and decreased lung compliance in BALB/c SCID mice. Further, IL-23 induced airspace inflammation and production of Th17-related cytokines and chemokines, while not impacting lung function in Rag2-/-γc-/- mice. This work suggests a novel role for IL-23 in airway disease as IL-23 itself could induce inflammation, which was predominately neutrophilic, in mice regardless of immune status. More significantly, IL-23 was also shown to modulate pulmonary expression of extracellular matrix-related proteins in these mice, which is currently under investigation.
Development of house dust mite-induced allergic airway disease in a murine model of sickle cell disease is associated with decreased regulatory cells and increased pulmonary pathology (HYP7P.283)
Authors: Adami Alexander , Bracken Sonali , Guernsey Linda , Andemariam Biree , Thrall Roger
Published: January 2023

The hematologic disorder sickle cell disease (SCD) is associated with unusually severe allergic asthma. To explore the mechanisms behind this heightened severity, allergic airway disease was induced in transgenic SCD mice and littermate (LM) controls via 2 wks of intranasal exposure to the clinically-relevant allergen house dust mite (HDM). Following final HDM exposure, animals were sacrificed for pathologic and cellular analyses. SCD was associated with nearly two-fold increase in leukocytes (p < 0.05) and more numerous inflammatory foci in the lung tissue as compared to LM controls. The proportion of CD4+Foxp3+ regulatory T cells was significantly reduced in SCD mice versus LM controls in the broncho-alveolar lavage (BAL) (9% vs. 14% of CD4+ T cells, p < 0.05) and lung (9% vs. 14% of CD4+ T cells, p < 0.01) but not in the hilar (lung-draining) lymph node (HLN). Furthermore, the proportion of CD5+ B cells in SCD mice was nearly half that of LM controls in the BAL (p < 0.0001), lung (p < 0.01), and HLN (p < 0.05). Intriguingly, this B cell population includes regulatory B cells that our laboratory has shown to play an important role in murine models of asthma. The dearth of regulatory cells in SCD may influence the increased pathology seen in the lungs of SCD mice and could contribute to the heightened severity of allergic asthma seen in human SCD. Treatment of asthma in SCD could be enhanced through efforts to target these cellular populations.
Silver nanoparticles impair human antimicrobial immune responses (INC6P.353)
Authors: Schwander Stephan , Zhang Junfeng (Jim) , Tetley Teresa , Gow Andrew , Chung Kian Fan , Ryan Mary , Porter Alexandra , Sarkar Srijata
Published: January 2023

Silver (Ag) nanoparticles (NPs) are widely used in consumer products potentially exposing humans via lungs, skin, digestive tract and insertion of medical devices. Therefore, biological risk assessments of Ag NPs are critical. We assessed the effect of Ag NPs [colloidal silver spheres of 20 nm (Ag20) and 110 nm diameters (Ag110) stabilized with either citrate or polyvinlypyrrolidone (PVP)] on human innate immune responses to Mycobacterium tuberculosis (M.tb), a respiratory pathogen that causes TB. Human monocyte-derived macrophages (MDMs), a model for alveolar macrophages, were exposed to multiple Ag NP doses for 4 hours. Ag NPs (at nontoxic concentrations, MTS assay) increased the expression of mRNA encoding IL8 (p<0.05) while decreasing the expression of mRNA encoding IL10 (p<0.05). Co-exposure of MDMs to Ag NPs and M.tb suppressed the M.tb-induced expression of mRNAs encoding IL1B, IL10, and TNFA in an Ag NP concentration-dependent manner (p<0.05). Ag20-citrate and Ag20-PVP showed stronger inhibitory activities than Ag110-citrate and Ag110-PVP. Ag NPs suppressed antimycobacterial immune responses by inhibiting NF-κB target genes including CSF2, CSF3, IL6, IL1A, IL1B and TNFA, components of the pathogen-induced TLR signaling pathway, crucial for innate antimycobacterial host immunity. These observations may be explained by the strong induction (p<0.05) of mRNA encoding Hsp72 following Ag NP exposure. Hsp72 can potentially inhibit M.tb-induced NF-κB activation.
Exocytosis of toxic primary granules by human neutrophils is associated with high surface TRPM2 expression and pinocytosis (INC6P.401)
Authors: Tirouvanziam Rabindra , Forrest Osric , Bowen James , Brown Milton , Ingersoll Sarah
Published: January 2023

Polymorphonuclear neutrophils (PMNs) play a key role in host defense against microbial infection. PMN responses culminate in the mobilization of highly toxic intracellular primary granules toward either the phagosome (during phagocytosis), nucleus (during NETosis), or plasma membrane (exocytosis). Mechanisms regulating the fate of PMN primary granules remain unclear. Here, we studied the dynamics of PMN primary granule exocytosis in two relevant sets of conditions: 1- in vivo migration of blood PMNs into chronically infected airways of patients with cystic fibrosis; 2- in vitro treatment of blood PMNs with latrunculin B and fMLF. Using flow and image cytometry, we show a striking resemblance between these two sets of conditions, with a close association between primary granule exocytosis (based on surface CD63 expression) and increased surface expression of the inflammatory calcium channel TRPM2. In addition, high primary granule exocytosis associates with major membrane rearrangements, as evidenced by the loss of surface phagocytic receptors (e.g., CD16 and CD35) and high pinocytosis (based on Lucifer Yellow uptake), a process previously linked to TRPM2 activation. All the while, exocytosing PMNs maintain their nuclear integrity. Thus, high primary granule exocytosis, surface TRPM2 upregulation and high pinocytosis are a concerted set of events indicative of a functional program in activated PMNs that is distinct from phagocytosis and NETosis.
AIM2 inflammasome is protective in regulating adaptive responses to oxidative stress in the liver (INM6P.410)
Authors: Sun Qian , Billiar Timothy , Scott Melanie
Published: January 2023

Inflammasome activation leads to activation of caspase1 and maturation of IL1β/IL18 in immune cells. Our previous data showed a novel role for caspase1 in hepatoprotection after redox stress by induction of mitochondrial autophagy, and this is independent of IL1β/IL18. Here, we further investigated inflammasome activation pathways after redox stress in liver. Mice deficient in known main inflammasome components NLRP1a, NLRP3, AIM2 or WT control (C57BL/6) mice underwent hemorrhagic shock with resuscitation (HS/R). NLRP3-/- mice had comparable IL18 levels, liver damage and hepatocyte (HC) caspase1-cleavage as WT mice at 6h. Knockdown of NLRP1a significantly suppressed levels of circulating IL-18 (162±21 vs 101±11pg/ml, p<0.01, control vs NLRP1a knockdown) after HS/R, but similar liver damage and HC caspase1 cleavage to control mice. AIM2-/- mice had similar levels of plasma IL18 as control mice (128±15 vs 135±29pg/ml), but significantly elevated liver injury (195±12 vs 995±287 IU/L, p<0.01) after HS/R, similarly to caspase1-/- mice. Similarly, in vitro AIM2-/- HC had decreased beclin1 expression (2.7 fold decrease in comparison with WT HC) and decreased mitochondrial clearance during redox stress as compared with WT cells. Taken together our data suggest that NLRP1-inflammasome regulates caspase1-activation in immune cells after redox stress, but that AIM2-inflammsome regulates caspase1-activation and mitophagy in HC/liver and is hepatoprotective after HS/R.
Reprogramming lipid metabolism primes host antiviral immunity (INM7P.431)
Authors: York Autumn , Argus Joseph , Zhen Anjie , Wu Nicholas , Sun Ren , Kitchen Scott , Bensinger Steven
Published: January 2023

Oxysterols, or oxidized derivatives of cholesterol, have emerged as important regulators of adaptive and innate immunity. However, the molecular mechanisms by which these lipids facilitate host defense remain indeterminate. To better understand how cellular sterol metabolism influences innate immunity, we have generated gain- and loss-of-function macrophages that have key components of sterol homeostasis perturbed. Remarkably, we find that genetic manipulation of sterol biosynthetic capacity, and cellular sterol homeostasis, intrinsically alters its innate immune program. Genetic inhibition of the sterol biosynthetic pathway renders cells intrinsically resistant to pathogen challenge. Conversely, ectopically enforcing a sterol synthetic program renders cells more susceptible to viral infections. Mechanistic studies indicate that disruption of sterol synthesis in macrophages activates an array of host defense pathways, most notably a robust type 1 IFN response and upregulation of the anti-viral proteins MX-1 and MX-2. Current studies are focused on defining how inhibition of the sterol biosynthetic pathway engages anti-viral immunity and determining if this response is broadly applicable to other cell types. These studies provide proof -of-principle evidence that direct manipulation of sterol homeostatic state of a cell is intimately associated with host defense and suggests that metabolic manipulation converts a cell from a permissive to resistant viral immune state.
Roles of viral inclusion bodies in innate immune evasion in infection with a novel bunyavirus (INM8P.441)
Authors: Xing Zheng , Wu Xiaodong , Qi Xian , Liang Mifang , Li Chuan , Cardona Carol , Li Dexin
Published: January 2023

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging febrile illness caused by a novel phlebovirus in the family Bunyaviridae. We have previously shown that innate immunity in host cells was modulated in infected cells and antiviral cytokine induction was suppressed by nonstructural protein NSs of SFTSV. In this study, we analyzed the pattern recognition receptors and the signaling pathways for mounting innate defense against viruses. Through studying virus-host interactions, we have explored the strategy exploited by SFTSV to evade host antiviral responses. We found that NSs can form inclusion bodies in infected cells. Viral inclusion bodies (IBs), which are accumulated aggregates of viral proteins, are commonly formed during viral replication in infected cells, but their role in viral immune evasion has barely been explored. Interestingly NSs interacted with multiple players in the signaling pathways. Through interacting with tank-binding kinase 1 (TBK1), NSs was able to sequester the IKK complex, including IKKε and IRF3 into IBs, albeit NSs did not interact with IKKε or IRF3 directly. In the presence of NSs, IRF3 could still be phosphorylated, but p-IRF3 was trapped into cytoplasmic IBs in SFTSV-infected cells; consequently, IFN-β induction was reduced and viral replication enhanced. Sequestering IKK complex, active IRF3, and other signaling molecules into viral IBs resulting in a reduced IFN response, is a novel mechanism for viruses to evade innate immunity.
MicroRNA-155 and MicroRNA-21 promote the expansion of functional myeloid-derived suppressor cells (IRC5P.473)
Authors: Li Limin , Zen Ke
Published: January 2023

Myeloid-derived suppressor cells (MDSC) are one of the main cell populations that negatively regulate immune responses. However, the mechanism underlying the expansion of MDSC remains unclear. Using miRNA microarray and TaqMan probe-based quantitative RT-PCR assay, we identified microRNA (miR)-155 and miR-21 as the two most upregulated miRNAs during the induction of MDSC from the bone marrow cells by GM-CSF and IL-6. High levels of miR-155 and miR-21 also were detected in bone marrow and spleen MDSC isolated from tumor-bearing mice. Our results also showed that TGF-b promoted the induction of MDSC through upregulating miR-155 and miR-21 expression. Overexpression of miR-155 and miR-21 enhanced whereas depletion of miR-155 and miR-21 reduced the frequencies of cytokine-induced MDSC. Subpopulation analysis indicated that miR-21 and miR-155 induced the expansion of both monocytic and granulocytic MDSC. Furthermore, miR-155 and miR-21 showed a synergistic effect on MDSC induction via targeting SHIP-1 and phosphatase and tensin homolog, respectively, leading to STAT3 activation. Finally, dexamethasone treatment strongly enhanced MDSC expansion through upregulating miR-155 and miR-21 expression, and the effect of dexamethasone on MDSC induction was abolished by depleting cellular miR-155 and miR-21. These results demonstrate a novel miR-155/miR-21-based regulatory mechanism that modulates functional MDSC induction.
IL-22-producing Th cells regulate myeloid-derived suppressor cells in Helicobacter pylori-associated immunopathogenesis (IRC5P.456)
Authors: Zou Quanming , Zhuang Yuan
Published: January 2023

Th cell responses are critical for the immunopathogenesis of gastritis induced by persistent infection with Helicobacter pylori (H. pylori). IL-22-producing Th cells represent a new T-cell subset, but their response, regulation, function, and clinical relevance in H. pylori-induced gastritis are presently unknown. We found that IL-22-producing Th cells accumulated in gastric mucosa of cytotoxin-associated Ag (CagA)+ H. pylori-infected patients and mice. CagA+ H. pylori-activated DCs secreted IL-23, which favored IL-22-producing Th cell polarization. IL-22-producing Th cell-derived IL-22 induced CXCL2 chemokine production by gastric epithelial cells that were induced to express IL-22R1 by CagA+ H. pylori, which in turn promoted CXCR2-dependent migration of myeloid-derived suppressor cells (MDSCs). Subsequently, IL-22 induced MDSCs to produce proinflammatory proteins S100A8 and S100A9, and MDSCs itself exerted immunosuppressive roles by suppressing Th1 cell response, in doing so, contribute to the development of H. pylori-associated gastritis. Collectively, this study identifies a novel regulatory network involving IL-22-producing Th cells, which in conjunction with DCs, gastric epithelial cells, and MDSCs exert an inflammatory and immunosuppressive mixed effect within H. pylori-induced gastric microenvironment. Efforts to inhibit IL-22-producing Th cell-MDSC pathway might therefore prove a valuable strategy in the therapy of H. pylori-associated gastritis.
A potential pathogenic role of myeloid derived suppressor cells in autoimmune arthritis (IRC5P.457)
Authors: Guo Chunqing , Hu Fanlei , Li Zhanguo , Wang Xiang-Yang
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitor cells and immature myeloid cells. Although the role of MDSCs in tumor immune evasion during cancer progression has received much attention, its involvement in other inflammatory autoimmune diseases, such as rheumatoid arthritis (RA) remains largely unknown. We show that the levels of MDSCs are significantly elevated in RA patients and during the progression of collage-induced arthritis (CIA) in mice. While MDSCs in arthritic mice are phenotypically distinct from neutrophils or inflammatory monocytes, they display strong T-cell suppressive activity in vitro, resembling the functionality of tumor expanded MDSCs. Surprisingly, the expansion of MDSCs in lymphoid organs or inflamed joints positively correlate with the disease severity in arthritic mice and RA patients. Additionally, MDSCs from arthritic mice or RA patients efficiently promote the differentiation of naïve CD4+ T cell precursors into T-helper 17 (Th17) cells. Furthermore, pharmacologic elimination or antibody depletion of MDSCs attenuates a Th17 response in arthritic mice, which concomitantly reduces articular cartilage damage and ameliorates the established disease. Our results suggest that an excessive accumulation of MDSCs may exacerbate autoimmune arthritic disease by promoting inflammatory Th17 cells. MDSCs may be exploited as a potential cellular target of intervention in autoimmune arthritis.
Metalloproteinase activity inhibits IL-23p19 expression in dendritic cells by targeting membrane stem cell factor affecting lung IL-17 response. (IRC5P.458)
Authors: Oriss Timothy , Krishnamoorthy Nandini , Raundhal Mahesh , Morse Christina , Chakraborty Krishnendu , Khare Anupriya , Huff Rachael , Ray Prabir , Ray Anuradha
Published: January 2023

Ligation of c-kit by membrane-bound stem cell factor (mSCF) leads to DC IL-6 production and a Th17 immune response, which also requires heterodimeric IL-23. However, the mechanisms regulating IL-23 production in DCs are not fully understood. We found that IL-23p19 gene expression in lung DCs is dependent on mSCF, which itself is regulated by metalloproteinase activity. Th1-inducing conditions enhanced protease activity causing cleavage of mSCF whereas the opposite was true for Th17-promoting conditions. In metalloproteinase-deficient mice, Th1-inducing conditions maintained mSCF expression thereby enhancing IL-23p19 in DCs. The functional consequence of this was enhanced IL-17 production in T cell/DC co-cultures in vitro, and increased numbers of IL-17-producing CD4+ T cells in the lung. Conversely, mSCF cleavage from bone marrow DCs in vitro by recombinant metalloproteinase led to reduced IL-23p19 expression under Th17-inducing conditions with dampening of intracellular AKT phosphorylation, a key component of the c-kit signaling pathway. Collectively, these results show that IL-23 production in DCs can be regulated by metalloproteinase activity on the c-kit/mSCF axis to control IL-17 production in the lung.
Metabolic mechanisms underlying IL-33 mediated Th2 polarization and allergic airway disease (IRM7P.494)
Authors: Lott Jeremy , Matta Benjamin , Mathews Lisa , Sperling Anne , Turnquist Heth
Published: January 2023

IL-33 is a recent IL-1 cytokine with emerging pleotropic functions. Experimental studies have suggested that IL-33 supports Treg responses, drives CD8+ T cell IFN-γ production, and acts as a potent mediator of Th2 responses. The current study tests the hypothesis that IL-33 modulates mDC gene expression and pathway activities facilitating Th2 polarization. Using gene expression and efficiency analysis, we identified multiple genes involved in iron homeostasis and metabolism which were upregulated in mDC by IL-33. Desferrioxamine (DFO) was utilized to assess the impact of blocking mDC iron uptake during IL-33 stimulation on subsequent Th2 polarization and capacity to stimuli allergic airway disease (AAD). Generation of CD4+IL-5+ Th2 cells by IL-33-exposed DC was ablated by iron chelation, resulting in decreased IL-5 production and cellular proliferation. However, induced Th1 responses were unaffected in DFO-treated LPS-stimulated DC. Using an OVA/IL-33 mDC induced model of AAD, blocking DC iron uptake with DFO decreased several phenotypic hallmarks of the disease, including IgE (p<0.05) and IL-33 production in the lungs of animals. In conclusion, iron uptake is necessary for IL-33 generation of pro-inflammatory DC supporting Th2 polarization. Blocking iron uptake in a model of mDC induced AAD alters disease phenotype when compared to control animals. These data suggest that regulation of iron uptake may be a potential mechanism behind IL-33 induction of Th2 responses.
A suppressive role of STAT1 in lupus-like chronic graft versus host disease (IRM7P.479)
Authors: Shao Wenhai , Lobue Monica , Gamero Ana , Cohen Philip
Published: January 2023

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease driven by genetic and environmental factors with unknown etiopathogenesis. The bm12→B6 chronic graft-versus-host disease (cGVHD) is a well-studied inducible mouse model of SLE. STAT1 is dynamically regulated during the course of immune and inflammatory responses. However, little is known about the role of STAT1 in autoimmune diseases. We induced cGVHD in B6.STAT1-KO and B6.WT mice by intraperitoneally injection of 1×108 splenocytes from bm12 mice. Mice were followed for autoimmune manifestations, such as mortality, skin rash, and renal involvement. Upon injection, WT mice developed anti-dsDNA autoantibodies starting at week 2 (as expected), with decline noted after week 4. In contrast, STAT1-deficient mice exhibited a prolonged and significant increase of anti-dsDNA autoantibody responses compared to WT mice (week 4 to week 8). Severe splenomegaly also developed in cGVHD STAT1-KO mice. Increased autoantibody titers were accompanied by increased proteinuria in mice lacking STAT1 due to immune complex deposition. Three months after injection, 40% of the STAT1-KO mice with cGVH disease were dead. Anti-dsDNA isotypes in cGVH diseased STAT1-KO mice were enhanced (IgG1 responses), but IgG3 responses were absent. STAT1 seems to suppress systemic autoimmunity by altering autoantibody isotype switching. Results from these studies may provide insights into the pathogenesis of STAT1 in SLE.
Production of class switched antibody secreting cells and antibody is inversely correlated with expression of the adaptor protein HSH2 (IRM8P.712)
Authors: Justement Louis , King R.
Published: January 2023

Control and HSH2 transgenic (HSH2-Tg) mice were compared to mice that express approximately 1/6th the normal level of HSH2 (HSH2-Lo) to determine if there is an inverse relationship between HSH2 expression and class switched antibody (Ab) production. In contrast to HSH2-Tg mice, HSH2-Lo mice have normal serum Ig titers for IgG1 and IgG2b and elevated titers of IgG3. HSH2-Lo mice also exhibit enhanced production of IgG1, and IgG2b, or IgG3 in response to challenge with either TD or TI Ag, respectively. Stimulation of B cells isolated from HSH2-Lo mice with anti-CD40 and IL-4 results in the production of significantly more antibody secreting cells (ASC) that produce IgG1 and IgE versus control B cells, whereas HSH2-Tg B cells produce significantly fewer class switched ASCs. Although HSH2 expression is associated with a modest effect on class switch recombination, changes in class switched ASC production are most closely linked to alterations in terminal differentiation of GC B cells into plasma cells. Terminal differentiation of class switched B cells from HSH2-Lo mice is significantly increased following in vitro stimulation, resulting in enhanced production of class switched ASCs compared to control B cells. Conversely, class switched B cells generated from HSH2-Tg mice exhibit a significant decrease in differentiation into ASCs in vitro. Thus, differential expression of HSH2 in the B lineage regulates multiple steps associated with the generation of class switched ASCs.