bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of organ-specific inflammation by CXCR5 during systemic autoimmunity (BA13P.131)
Authors: Sharma Rahul , Abaya Christian , Sharma Poonam , Stremska Marta , Jose Sheethal
Published: January 2023

Mice and humans deficient in the Foxp3+ T regulatory cells (Tregs) develop systemic autoimmunity affecting multiple organs and die prematurely. Mice deficient for interleukin (IL)-2 (IL2KO) have reduced Tregs and develop inflammation mainly in liver, colon, pancreas and salivary glands. We found that the CD4+ T-cells in the secondary lymphoid organs IL2KO mice highly express CXCR5, a chemokine receptor and a marker for T-follicular helper (TfH) cells. We hypothesized that CXCR5 expression may regulate inflammation by regulating T-cell recruitment and organ-specific autoantibody production. We found that CXCR5+CD4+ T-cells were highly enriched in these inflamed organs of IL2KO mice. Rendering the IL2KO mice deficient for CXCR5 led to prolonged lifespan compared to CXCR5-sufficient IL2KO mice and protection against inflammation especially in liver and colon and partial protection against inflammation in pancreas and salivary glands. This was accompanied with a reduction of activated B and T cells in the secondary lymphoid organs as well as reduced production of inflammatory cytokines by the T-cells. However, no significant correlation was observed between CXCR5 expression and antibodies to liver and pancreatic auto-antigens. The data suggests that CXCR5 expression regulates organ-specific inflammation during Treg-deficiency -induced systemic autoimmunity, through regulation of T-cell trafficking, with an undetectable contribution to autoantibody production.
Novel function of ECM1 on inhibiting Th17 differentiation and infiltration in EAE development (BA13P.116)
Authors: Sun Bing , Su Pan , Zhou Haiyan , Zhang Yaguang
Published: January 2023

Our previous work has demonstrated that Extra-cellular Matrix Protein 1 (ECM1) is a specific secreted protein by Th2 cells (Nature Immunology, 2011). In this study, we demonstrated that ECM1 protein is capable of inhibiting Th17-type cell differentiation during EAE initiation and then prevents EAE development. The mechanisms study showed that ECM1 suppresses TGF-b maturation and in turn to inhibit Th17-type cell differentiation. It is also observed that ECM1 can inhibit Th17 cell infiltration into neuron central system through targeting MMP9. Our results indicate that ECM1 has a novel function to suppress Th17 cell differentiation and infiltration in EAE mice model.
Disruption of maternal tolerance during pregnancy leads to Treg repopulation of the antigenic UPI (BA13P.123)
Authors: Collier Ai-ris , Snead Andrea , Cook Heather , Yesayan Maria , Kahn Daniel
Published: January 2023

Background Regulatory T cells (Treg) are essential for normal pregnancy outcome. In vivo, Treg depletion during pregnancy leads to autoimmunity that results in total pregnancy loss. The paucity of Treg and effector cells in normal pregnancy uteroplacental interfaces (UPI) renders the mechanism unclear. Methods Low-dose (0.1ug) diphtheria toxin was given on GD11 to female C57-Bl6 FoxP3-DTR under a syngeneic pregnancy. On day 17, pup weight and gender was determined and UPI were removed and subject to FACS and confocal immunofluorescence. Results Subtle Treg depletion during pregnancy resulted in effector T cell influx preferentially to UPIs of antigenic (male) fetuses resulting in a reduction of fetal weights. Surprisingly, we also observed an increase of Treg numbers in the antigenic UPIs. Preliminary results indicate a local interaction between antigen-presenting cells, T cells and Treg in the previously depleted UPI. Conclusions Systemic interruption of Treg tolerance during pregnancy allows for maternal attack on antigenic fetuses. In the unperturbed UPI, the absence of Treg suggests that maternal tolerance is mediated elsewhere. In disrupted maternal tolerance, the repopulation of Treg to the UPI provides insight into Treg cellular interactions that may contribute to the re-establishment of tolerance under physiologic conditions.
The type 1 diabetes resistance locus B10 Idd9.3 impairs B cell development and implicates microRNA-34a as a regulator of T1D pathogenesis (BA14P.200)
Authors: Waldner Hanspeter , Berry Gregory , Budgeon Lynn , Cooper Timothy , Christensen Neil
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease resulting from the T cell-mediated destruction of insulin-producing pancreatic beta cells. Multiple insulin-dependent diabetes (Idd) loci confer T1D susceptibility or resistance in both humans and nonobese diabetic (NOD) mice, but the genes and mechanisms by which they control disease development are largely unknown. NOD.B10 Idd9.3 mice contain the Idd9.3 locus from T1D-resistant C57BL/10 mice that encodes 19 genes, including microRNA (miR)-34a, and confers partial T1D protection. B cells have been shown to play a critical role in priming autoantigen-specific CD4+ T cells in NOD mice. We show that early B cell development is impaired in NOD.B10 Idd9.3 mice, resulting in significant reduction of splenic B cells compared to NOD mice. Molecular analysis revealed that miR-34a expression was significantly elevated in B cell progenitors and marginal zone B cells of NOD.B10 Idd9.3 mice compared to NOD mice. Furthermore, miR-34a expression inversely correlated with levels of Foxp1, an essential regulator of B cell lymphopoiesis, which is directly repressed by miR-34a. Importantly, peripheral B cell paucity in NOD.B10 Idd9.3 mice was associated with impaired proliferation of islet-antigen-specific CD4+ T cells in response to endogenous autoantigen in pancreatic lymph nodes. These data suggest that miR-34a negatively regulates B cell development in NOD.B10 Idd9.3 mice and thereby may contribute to their T1D protection.
Induced pancreatic inflammation is controlled in mice by extrapancreatic insulin expression and CD8 T cells limiting autoimmune attack (BA14P.209)
Authors: Girtman Megan , Pavelko Kevin , Pease Larry
Published: January 2023

Type 1 diabetes can be induced by virus expressing SIINFEKL in Rat Isulin Promoter (RIP)-OVA transgenic mice harboring adoptively transferred naïve antigen-specific OT1 T cells. By titrating the number of transferred OT-1 cells, disease outcome can be manipulated. Mice receiving high numbers of OT-1 cells develop autoimmunity and rapidly succumb to disease. Mice receiving limiting numbers develop hyperglycemia transiently and then over 10 days recover glucose homeostasis. Recovered mice exhibit restored normal resting blood sugar levels and are able to regulate glucose during a stress test. Histological examination of the pancreas showed extensive pancreatic destruction with no or very few remaining insulin-positive islets. Using RIP-OVA mice expressing luciferase under the mouse insulin promoter, pancreatic insulin promoter activity was extinguished, while liver expression increased in recovered animals, suggesting an ectopic source of insulin can support glucose homeostasis in mice with extensively damaged islets. Pancreatic infiltrates during peak inflammation contained host-derived CD8 T cells in addition to the infiltrating OT-1 cells. These host CD8 T cells were absent in CD8 deficient RIP-OVA mice, and diabetes occurred with fewer transferred OT-1 cells and was quickly fatal. Reconstitution of the CD8 compartment protected these animals. These findings indicate the importance of host CD8 T cells in limiting autoreactive T cell-mediated destruction of islets.
PU.1-dependent T cell function limits inflammation in EAE (BA14P.214)
Authors: Awe Olufolakemi , Pham Duy , Kaplan Mark
Published: January 2023

T helper cell subsets mediate autoimmune disease through the secretion of specific cytokines that recruit other immune cells and alter the inflammatory microenvironment. IL-9-secreting Th9 cells can contribute to the development of autoimmune diseases, including Experimental Autoimmune Encephalomyelitis (EAE), although the mechanisms are unclear. Elimination of IL-9 or IL-9R in models of EAE has led to somewhat conflicting results. To attempt a different approach to this question we examined the development of EAE in mice with a T cell-specific deletion of the Sfpi1 gene, which encodes PU.1, a transcription factor important for the production of IL-9. Interestingly, Sfpi1lck-/- mice had significantly worse disease scores compared to WT mice beginning 19 days after initial MOG35-55 immunization. The disease scores for the Sfpi1lck-/- mice persisted through day 30 in contrast to disease resolution in WT mice. Although few IL-9 secreting T cells were found among CD4+ T-cells isolated from the brains of Sfpi1lck-/- mice 25 days after initial immunization, there were significantly higher frequencies of IFN-γ, IL-17a, and IFN-γ+IL-17a+ when assayed by intracellular staining. There was also higher MOG-specific total IgG and IgG1 levels in the Sfpi1lck-/- mice. These results suggest that PU.1 dependent immunity limits autoimmune inflammation, particularly during the resolution phase of disease.
Increased lymph node CD8+ T cell proliferation and memory cell markers upon conditional ablation of Langerhans cells in autoimmune mice (BA14P.215)
Authors: Gutierrez Miguel-Angel , Halder Ramesh , Valera Isela , Singh Ram
Published: January 2023

Systemic autoimmune diseases such as lupus are characterized by inflammation in many organs. The role of local tissue-resident immune cells in specific organ involvement in these diseases remains unclear. We evaluated the role of Langerhans cells (LC) as prototypic tissue-resident dendritic cells (DC) in eliciting local immune defects in the MRL model of lupus. Injection of diphtheria toxin (DT) in Lang-eGFP.DTR knock-in mice results in depletion of langerin+ dermal DC (dDC) and LC. To assess LC’s role in systemic autoimmunity, we introgressed the knock-in mutation from the stock B6 onto the MRL background. Serial DT injections every 7-10 days exacerbate inflammation in the skin (King et al) and cornea in MRL but not in B6 mice. To investigate underlying mechanisms, we assessed the effect of LC depletion on T cells in skin/eye draining cervical lymph nodes (cLN). There was a significantly higher proliferation of CD8+, but not CD4+, T cells in cLN of LC-ablated than control mice; such effect was not seen in spleen CD8+ T cells. Also, LC-ablated MRL, but not B6 mice, exhibited reduced CD62L on cLN T cells. Further, freshly isolated dDC were more efficient than LC in promoting the proliferation of CD8+, but not CD4+, cNL T cells. Such local regulation of CD8+ T cells may play a role in immune tolerance locally. A defect in this mechanism owing to reduced LC migration in MRL mice (Eriksson and Singh, 2008) may confer skin/eye inflammation, without affecting other organs
Foxp3-mediated inhibition of Akt inhibits Glut1 expression in human T regulatory cells (BA14P.203)
Authors: Basu Samik , Bowen Britany , Shevach Ethan
Published: January 2023

Thymic-derived CD4+CD25+Foxp3+ T regulatory cells (Tregs) have a diminished capacity to activate the PI3K/Akt pathway. Although blunted Akt activity is necessary to maintain Treg suppressive function, other consequences of decreased Akt signaling are unclear. Glucose transporter 1 (Glut1) is a cell surface receptor responsible for facilitating glucose transport across plasma membranes and expression of Glut1 is tightly coupled to co-stimulatory signals and Akt phosphorylation. Freshly isolated human Tregs were unable to up-regulate Glut1 in response to TCR and co-stimulatory signals when compared to similarly activated effector T cells. Furthermore, stimulated Tregs utilized less glucose than stimulated effector T cells in ex vivo cultures. Introduction of Foxp3 into effector T cells by lentiviral-mediated transduction inhibited not only Akt activation, but also Glut1 expression, indicating that Foxp3 can regulate Glut1. Together, these results illustrate the molecular basis underlying differential glucose metabolism between human Tregs and effector T cells as well as revealing potential pathways for immune modulation.
T cell movement into the pancreas based on insulin receptor expression (BA14P.208)
Authors: Al-Dieri Ali , Esakov Emily , Slotterbeck Brandon , McInerney Marcia
Published: January 2023

In the nonobese diabetic (NOD) mouse, type 1 diabetes is an autoimmune disease characterized by insulitis and T cell-mediated beta cell destruction. The importance of insulin receptor (IR) expression in the pathogenesis of diabetes was examined since IR is a chemotactic molecule capable of directing cell movement in response to a gradient of insulin. Our published data shows that T cells, obtained from NOD diabetic mice, expressing a high density of IR can aggressively transfer insulitis and diabetes while T cells with low to negative amounts of IR, are capable of neither. Therefore, IR may function to deliver T cells to the islet where Ag specific cells within the population can recognize and destroy beta cells. Recently, it has been shown in NOD mice that benign insulitis can occur in the absence of T cell-MHC/Ag interactions. The object of this project was to test whether T cells expressing IR, can move into the pancreas based on insulin chemotaxis alone, in normal strains of mice. A transgenic C57Bl/6 mouse was made in which the mouse IR gene with a 3XFLAG tag was placed behind the CD3 promoter and enhancer to target IR expression on all T cells. In this transgenic model there is IR expression on all T cells and with age the transgenic mice exhibit insulitis, but not diabetes. Distinct areas of the IR mediate metabolic or chemotactic signaling indicating that a specific part of the IR may be selectively targeted for therapeutic intervention to block T cell movement.
Delineating the mechanisms of immunomodulatory effects of sex steroids in autoreactive T cells (BA15P.219)
Authors: Jia Ting , Massilamany Chandirasegaran , Reddy Jay
Published: January 2023

SJL mice immunized with proteolipid protein (PLP) 139-151 show a gender disparity in the development of autoimmune encephalomyelitis in that only females show a chronic relapsing-remitting type of paralysis, but their underlying mechanisms are unknown. We tested the hypothesis that sex steroids differentially regulate the expansion of autoreactive T cells. To this end, we have established an in vitro system to determine the effects of sex steroids on the expansion and functions of antigen-specific T cells based on proliferation assay, major histocompatibility complex class II dextramer staining, cytokine analysis and cell viability assays. Regardless of gender, we noted that the T cell responses to PLP 139-151 were significantly lower in cells exposed to either testosterone or estradiol as compared to vehicle-treated cultures, but the threshold for hormone-sensitivity is two-fold higher in males than females. Importantly, the reduced proliferative responses were found not to be due to hormonal inhibition, rather, the hormones promoted cell death. Conversely, while, the hormones reduced the number of interferon-gamma-producing cells in both the genders, interleukin (IL)-17-producing cells were reduced only in females; whereas other cytokines, such as IL-2, IL-4, IL-10, and IL-22 remained unaltered. Taken together, the data suggest that the immunomodulatory effects of sex steroids involve multiple pathways, and cell death may be the primary underlying mechanism.
Genetic deletion of S100A9 fails to abrogate psoriasiform inflammation in the KC-Tie2 mouse model due to IL-12/23 compensation. (CAM5P.237)
Authors: Ward Nicole , Fritz Yi , Dawes Sean , Diaconu Doina , Golden Jackelyn , Wang Yunmei , Simon Daniel , McCormick Thomas
Published: January 2023

The proinflammatory proteins S100A8/9 are purported to play a pathogenic role in psoriasis. A recent report showed genetic deletion of S100A9 strongly attenuated ear skin inflammation in the K5-JunB:Jun mouse and imiquimod skin inflammation models. We previously reported increased expression of S100A8/9 (>135-fold; P<0.05) in the KC-Tie2 psoriasis mouse model, with expression downregulated following disease reversal. We sought to confirm the importance of S100A9 in disease pathogenesis and mated KC-Tie2 mice with S100A9 KO mice. In contrast to recent reports, KC-Tie2-S100A9 KO mice (n=11) had similar levels of skin inflammation (acanthosis), T cell and myeloid cell skin-infiltration as KC-Tie2 mice (n=9); despite significantly decreased levels of DefB4, IL-1α, IL-17C and TNFα (all P<0.05). Interestingly, KC-Tie2-S100A9 KO animals had significantly elevated splenic and skin-draining CD11b+Ly-6C+ pro-inflammatory monocytes (27% and 86%, respectively; P<0.05) compared to KC-Tie2 animals, increased expression of skin IL-12/23 and IL-17A/F and sustained levels of skin IFNγ, IL-22, IL-6 and KLK6, potentially explaining the sustained presence of disease. These results suggest that the IL-23-Th17 inflammatory axis may compensate for genetic ablation of S100A9 and sustain psoriasiform skin inflammation in KC-Tie2 animals. Thus, the role of S100A9 in skin inflammation may be model dependent and further investigation is needed to establish S100A9 as a therapeutic target in psoriasis.
SOCS proteins regulate insulin receptor substrate (IRS)-2 activation in response to IL-4 and 13 in monocytes and macrophages (CCR5P.247)
Authors: McCormick Sarah , Gowda Nagaraj , Fang Jessie , Heller Nicola
Published: January 2023

Allergic asthma is a chronic lung disease driven by Th2 cytokines IL-4 and -13. In macrophages, IL-4/13 bind IL-4 receptors, which signal through IRS-2/STAT6, inducing M2 differentiation. We found that IL-4/13 stimulation of human monocytes/mouse macrophages leads to rapid tyrosine phosphorylation (pY) of IRS-2, peaking at 60min and returning to baseline by 150min. Mechanisms that regulate IRS-2 activity and M2 differentiation are not understood. Based on rapid de-phosphorylation of IRS-2, we hypothesized that SOCS proteins regulate IL-4/13 induced IRS-2 activation, thereby limiting M2s. To address this, we tracked the kinetics of SOCS induction following IL-4/13 exposure and found that SOCS1 and CIS are highly induced in human monocytes and mouse macrophages, respectively, while SOCS3 is only moderately induced. By co-immunoprecipitation, we found that SOCS1 and CIS complex with IRS-2 in response to IL-4/13. Furthermore, we found that siRNA knockdown of SOCS1 and CIS prolonged IRS-2 pY and enhanced human and mouse M2 differentiation respectively. Since SOCS proteins complex with ubiquitin ligases to promote proteasomal degradation, we examined the effect of proteasome inhibitors on IL-4/13-induced IRS-2 pY. Proteasome inhibition prolonged IRS-2 pY and enhanced M2 gene expression. These findings identify SOCS1 in human and CIS in mouse macrophages as critical negative regulators of IL-4/13-induced IRS-2 activity/M2 differentiation and are novel therapeutic targets for asthma.
Activator of G-protein signaling 3 (AGS3) binds GRK6 via its G-protein (GPR) domain to modulate CXCR2 mediated cellular responses. (CCR5P.261)
Authors: Singh Vandana , Smith Nikia , Richardson Ricardo
Published: January 2023

AGS3, a receptor-independent activator of G-protein signaling, comprises of seven tetratricopeptide (TPR) motifs and four GPR motifs joined by a linker sequence. Previous studies have demonstrated that TPR domain of AGS3 is important for determining the positioning of AGS3 within the cell, whereas, GPR domain serves as a docking site for Gαi-GDP leading to AGS3-Gαi signaling. We have recently shown that GRK6 complexes with AGS3-Gαi to regulate CXCR2-mediated cellular functions including Ca2+ mobilization, mitogen-activated protein kinase (MAPK) activation, receptor desensitization and down-regulation. In the current study mutants from the GPR and TPR domains were used to determine which motif(s) of AGS3 is (are) involved in binding with GRK6. Co-imunoprecipitation studies in RBL cells co-transfected with wild type and mutants AGS3 showed that, upon stimulation with interleukin 8 (IL-8/CXCL8), GRK6 interacts with the GPR domain of AGS3. Overexpression of AGS3 and the GPRs motif (ΔTPR7-link), but not the TPRs motif (TPR), in RBL-CXCR2 inhibited CXCL8-induced intracellular Ca2+ mobilization and MAPK activation. Taken together, these results indicated that AGS3 binds to GRK6 via the GPR domain to modulate CXCR2-mediated signal transduction.
FcγRIIB-mediated dissociation of BCR-CD19 microclusters and hyperreactivity of lupus B cells (HUM7P.303)
Authors: Liu Wanli , Xu LiLing
Published: January 2023

B cell activation is regulated through the interplay between the B cell receptor (BCR) and the inhibitory co-receptor FcγRIIB or activating co-receptor CD19. Recent studies suggest that the BCR microcluster is converted to a signaling active state upon its colocalization with CD19 microclusters to activate B cells. Here using TIRF microscopy-based live cell imaging techniques, we show that FcγRIIB can inhibit B cell activation by a previously uncharacterized mechanism of blocking the spatial-temporal colocalization of BCR and CD19 microclusters within the immunological synapse (IS). This inhibitory function of FcγRIIB mainly relies on its transmembrane domain (TM). Indeed, human primary B cells from SLE patients homozygous for the gene encoding FcγRIIB-I232T lose the ability to block the synaptic colocalization of BCR with CD19, leading to the dis-regulated recruitment of pCD19, pSyk and pPI3K to membrane proximal signalosome. Furthermore, FcγRIIB-I232T homozygous SLE patients are more likely to develop severe clinical symptoms characterized by high anti-ds DNA antibody titers and the involvement of kidney, central nervous system, gastrointestinal tract, lung and heart compared to patients carrying either one or both WT copies of FcγRIIB. These observations may explain the hyper-reactive features of SLE patient B cells and provide a potential target through FcγRIIB for the prophylactic diagnosis, evaluation of disease progression and potential therapies.
Inflammatory responses in myopia progression (HUM7P.317)
Authors: Wan Lei , Lin Hui-Ju , Hsu Yu-An
Published: January 2023

Myopia is an important and common eye disease as it can induce many other associated eye diseases and even lead to legal blindness. The prevention and treatment of myopia is an important public problem worldwide. We found that the expression levels of several inflammatory molecules are altered in the retina and sclera of FDM hamsters. To affirm the possible correlation between inflammatory reaction and myopia progression, cyclosporine A was used to treat FDM rats. We found that inhibiting inflammatory responses in the eye slowed myopia progression, whereas increasing inflammation by LPS and PGN enhanced myopia advancement. The expression levels of TGF-β, MMP2, CHRM1, CHRM3, Id1, c-Fos, NF-κB, IL6, and TNF-α were up-regulated in occluded eyes and down-regulated when treated with atropine and cyclosporine A. Id1, c-Fos, NF-κB, IL6, and TNF-α are known to have important roles in inflammatory reactions. Our results showed that immune and inflammatory reactions would alter the progression of myopia through modulation of inflammation responses.
An iNKT cell expansion causes premature delivery in mice (HUM7P.323)
Authors: Stlouis Derek , Sanchez-Rodriguez Elly , Milovic Tatjana , Romero Roberto , Gomez-Lopez Nardhy
Published: January 2023

Objective: Maternal-fetal tolerance involves a tight balance between the innate and adaptive immunity, which avoids rejection of the semi-allogeneic embryo. The innate and adaptive limbs of the immune response are bridged by iNKT cells; therefore, we hypothesized that an alteration in their number and/or activation breaks maternal-fetal tolerance, leading to premature delivery. Methods: Pregnant mice were injected on E16.5 with either α-galactosylceramide (an iNKT activator; 2 μg) or PBS as a control (n=19 each). Gestational length and the rates of stillbirth and long-term pup survival were recorded. Peripheral blood, decidua, liver, spleen and lymph nodes were collected just prior to delivery from additional treated and control mice (n=8-9 each). In tissue cell suspensions, iNKT phenotype and activation were analyzed by flow cytometry. Results: 1) iNKT activator treatment promoted premature delivery (E18.09±0.60; controls delivered on E19.51±0.78); 2) iNKT activator treatment increased the rate of stillbirth (63.63 vs. 21.94%); 3) iNKT activator treatment did not reduce long-term pup survival; 4) iNKT activator treatment caused an expansion of iNKT cells (CD1d tetramer+NK1.1+CD49b+) in peripheral blood, liver and decidua, but not in spleen and lymph nodes; 5) iNKT cell activation was unchanged by iNKT activator treatment. Conclusion: Treatment with α-galactosylceramide causes an expansion of systemic and local iNKT cells leading to premature delivery in mice.
Examining a potential link between antigen-presentation and metabolic dysregulation (HUM7P.289)
Authors: Bhagwandin Candida , Whalen Michael , Ishido Satoshi , Szajman Adam , Truong Sarah , Tsao Tsu-Shuen , Lybarger Lonnie
Published: January 2023

Antigen-presentation is induced following interaction of an antigen-presenting cell (APC) with stimuli, including microbial ligands and cytokines, whereupon Major Histocompatibility Complex II (MHC-II) and co-stimulatory molecule, CD86, surface expression is significantly upregulated. At the surface of the APC, the immune modulators provide signals critical for T cell activation and induction of the appropriate adaptive immune response. As key regulators of the T cell-APC conversation, it is imperative to gain a thorough understanding of the underlying mechanisms regulating expression and function of antigen-presenting molecules, and the biological consequences of dysregulation. Indeed, our lab has demonstrated that Membrane-Associated RING-CH1 (MARCH1), a highly-conserved E3 ubiquitin ligase identified as a potent regulator of both MHCII and CD86 in APCs, is stringently regulated at multiple steps, indicating a need for precise control of antigen-presentation. Interestingly, MARCH1-deficient and hemizygous mice show signs of mild hyperglycemia, an increase in the visceral adipose tissue (VAT), and increased inflammation of the VAT, all characteristic of insulin-resistance and type II diabetes progression. Collectively, the data support the hypothesis that antigen-presentation is tightly regulated to ensure a rapid adaptive immune response, and suggests a novel role for components of this pathway in the maintenance of insulin sensitivity in healthy adipose tissues.
In vitro hyporesponsiveness of CD4+ and CD8+ T cells in septic patients with faecal peritonitis (HUM8P.326)
Authors: Gore David , Preciado-Llanes Lorena , Mills Gary , Heath Andrew , Read Robert
Published: January 2023

The immunological processes behind sepsis are ill defined. Predicting or diagnosing sepsis, based on cellular immune parameters is a research priority. This study quantified differences in peripheral blood mononuclear cell (PBMC) and cytokine levels between septic patients with fecal peritonitis, matched surgical patients (no sepsis) and healthy controls. To assess function, PBMC were stimulated in vitro with anti-CD3±anti-CD28 antibodies. Four days later T and B cell proliferation were measured by CFSE and activation by expression of CD25 and CD69 on T cells and CD86 on B cells. Viability was assessed by flow cytometry. Concentrations of a range of cytokines were determined from plasma and supernatants (post-stimulation) by multiplex cytometric bead array. On day of phlebotomy there were reduced CD4+ and increased CD19+ cells in septic patients. Plasma IL-6 and IL-8 levels were higher in septic patients, IFN-γ and IL-13 were lower. There was reduced proliferation and activation of T cells from septic patients in response to anti-CD3+anti-CD28. Supernatant levels of IFN-γ, TNF-α and interleukins 1β, 5, 6, 13 and 17A were lower in septic patient samples. There were no initial differences in PBMC viability, but septic patient PBMC had reduced survival on incubation. In summary, we found reduced T cell numbers and a functional impairment of remaining T cells in septic patients.To our knowledge this is the first report of an immune function assay across these three groups.
Intensive immune suppression of Bolivian squirrel monkeys activates latent polyomavirus (HUM8P.337)
Authors: Nehete Pramod , Ruiz Julio , Nehete Bharti , Vanchiere John
Published: January 2023

The squirrel monkey polyomavirus (SMPyV) is commonly found in Bolivian squirrel monkeys, and, as such, the squirrel monkey may be a useful model for the study of polyomavirus-associated pathogenesis and experimental treatment and prevention strategies. Squirrel monkeys were given single or multiple doses of an anti-B cell antibody (Rituximab) and/or an anti-CD8 monoclonal antibody (7pt-3F9) which resulted in a complete CD20+lymphocyte depletion and transient CD8+lymphocyte depletion as compared to control animals. The animals remained clinically well, suggesting that the intensity and/or duration of immune suppression were inadequate to trigger pathogenic reactivation of the latent polyoma and herpes viruses. Quantitative PCR assays revealed excretion of the SMPyV in the urine within one week after the 7pt-3F9 infusion, continuing until the end of the study. At necropsy, spleen, liver, lymph nodes, cerebrum, cerebellum and kidney were positive for SMPyV DNA. Histologic and immunohistochemistry studies revealed moderately increased numbers of meningeal cells and inflammatory mononuclear cells in the kidney, renal medulla and foci of increased Ki67 staining in distal tubular epithelial cells in antibody treated monkeys compared to untreated monkey. One animal had multifocal encephalitis with prominent CD3+lymphocyte collections throughout the cortex. These results suggest that the transient depletion of CD8+lymphocytes may be a useful trigger for SMPyV shedding.
Multi-drug resistant Th17 cells: new players in autoimmune and steroid-resistant inflammation (HUM8P.351)
Authors: Sundrud Mark , Ramesh Radha , Kozhaya Lina , McKevitt Kelly , Djuretic Ivana , Carlson Thaddeus , Quintero Maria , McCauley Jacob , Abreu Maria
Published: January 2023

Th17 cells are generally regarded as key effectors of autoimmune disease. However, not all Th17 cells are pro-inflammatory. Furthermore, the defining features of "pathogenic" Th17 cells in humans are unknown. We have found that pro-inflammatory human Th17 cells are restricted to a subset of CCR6+ CXCR3(hi) CCR4(lo) CCR10- CD161+ T cells that expresses the drug transporter multi-drug resistance-1. Unlike MDR1- human memory T cell subsets, MDR1+ Th17 cells produce both Th17 and Th1 cytokines upon stimulation; do not express IL-10 or other anti-inflammatory molecules; express high levels of IL23R as well as other genes selectively expressed by pathogenic mouse Th17 cells; and display heightened responsiveness to IL-23 stimulation. In vivo, MDR1+ Th17 cells accumulate in the gut of patients with active Crohn's Disease, where they display a marked pro-inflammatory transcriptional signature relative to local MDR1- effector T cells. In addition, MDR1+ Th17 cells are refractory to the immunosuppressive effects of several steroids used to treat autoimmune disease in the clinic. Thus, MDR1+ Th17 cells may be important mediators of autoimmune inflammation, particularly in clinical settings of steroid-resistant disease.
Plasma chemokine as potencial biomarker of cardiac morbidity in Chagas disease (HUM8P.330)
Authors: Sousa Giovane , Gomes Juliana , Damásio Marcos Paulo , Fares Rafaelle , Chaves Ana , Ferreira Karine , Medeiros Nayara , Nunes Maria do Carmo , Corrêa-Oliveira Rodrigo , Rocha Manoel Otávio
Published: January 2023

The immune response appears to be associated with morbidity in Chagas disease. The aim of this study was to evaluate the plasma chemokine levels in well-defined clinical polar groups of chagasic patients and its relationship with morbidity. Patients infected with Trypanosoma cruzi were grouped as indeterminate (IND) and cardiac (CARD) forms ranging from 23 to 69 years of age. The IND group included 82 individuals without cardiac involvement, while the CARD group included 94 patients presenting dilated cardiomyopathy. Healthy non-chagasic individuals were included as a control group (NI). The plasma chemokine levels, such as inflammatory protein 10 (IP-10), monocyte chemoattractant protein (MCP) 1 and monokine induced by interferon gamma (MIG), proved to be the highest in the CARD group. However, no significant difference could be observed when comparing the RANTES expression in the IND and CARD groups. Correlation analysis showed that higher IP-10, MCP-1 and MIG expression was associated with worst cardiac function, as determined by left ventricular ejection fraction and left ventricular diastolic diameter values. The findings of this study indicate that a fine balance between homeostatic and inflammatory chemokines might represent a key element in the establishment of the distinct forms of chronic Chagas disease. Moreover, this inference involves important implications to better understand the pathogenesis and clinical management of patients with Chagas disease.
Susceptibility of NOD/scid IL-2Rγnull mice inoculated with alcoholic patient white blood cells to the pulmonary infection of Klebsiella pneumoniae (HUM8P.331)
Authors: Ito Ichiaki , Asai Akira , Ohama Hideko , Tsuchimoto Yusuke , Tsuda Yasuhiro , Higuchi Kazuhide , Nakamura Kiwamu , Suzuki Sumihiro , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Alcoholics are known to be very susceptible to various opportunistic infections. In this study, host antibacterial resistance of alcoholics to K. pneumoniae infection was characterized in a murine chimera model of alcoholics. A chimera model of alcoholics (patient chimeras) was created in NOD/scid IL-2Rγnull mice irradiated with γ-rays (3.5 Gy) after inoculation with WBC (5 x 106 cells) from alcoholic patients. As a control (healthy chimeras), the same scid mice were inoculated with 5 x 106 cells of healthy donor WBC. Both chimeras were intratracheally infected with K. pneumoniae (100 CFU). In the results, bacterial growth was detected in the lungs and kidneys of patient chimeras, while significant numbers of bacteria were not isolated from these organs of healthy chimeras. All patient chimeras died within 2 days of infection, while 75% of healthy chimeras survived more than 7 days after the infection. Alcoholic patient CD14+ cells killed K. pneumoniae, while the pathogen was not killed by the same cells isolated from healthy donors. CD14+ cells isolated from WBC of alcoholic patients were shown to be IL-10-producing CCL1+CD163+ cells (M2bMφ), while these cells were not isolated from WBC of healthy donors. These results indicate that host antibacterial resistance is not displayed in humanized chimeras created with alcoholic patient WBC. M2bMφ demonstrated in alcoholic patient WBC may play a role on the impaired resistance of alcoholics to opportunistic infections.
Infection-induced preterm delivery does not involve macrophage M2-M1 polarization (HUM8P.333)
Authors: Gomez-Lopez Nardhy , Mial Tara , Robertson Sarah
Published: January 2023

Objective: Macrophages seem to have a regulatory phenotype (M2) during pregnancy since their depletion causes preterm delivery in mice (Gomez-Lopez et al, unpublished data). Our aims were to investigate whether there is macrophage M2-M1 polarization during infection-induced preterm delivery and to elucidate whether this polarization is associated with T cell phenotype changes. Methods: Pregnant (16.5 post-coitum) FVB/NJ mice were injected intraperitoneally with either 30µg/200µL of LPS (E. coli 0111:B4) or 200µL of 1X PBS (n=10-11 each). The mice were euthanized 12 hours after injection, prior to delivery. Peripheral blood, decidua, uterus, placenta, and fetal liver were harvested into cell suspensions. The proportion of M1and M2 macrophage populations, CD4+ and CD8+ T-bet+ cells, Th17 cells and CD4+ and CD8+ Tregs were analyzed by flow cytometry. Results: 1) There was no macrophage M2-M1 polarization in maternal or fetal tissues from mice treated with LPS, 2) Uterine and decidual macrophages were fewer in LPS treated mice than in controls, 3) CD4:CD8 T cell ratio in peripheral blood was altered with LPS treatment, 4) Circulating CD3+CD4+Tbet+ (Th1) cells were greater in LPS treated mice than in controls, and 5) No changes were observed in fetal tissues from LPS treated mice. Conclusions: Endotoxin treatment reduces the proportion of macrophages and increases circulating Th1 cells, but it does not cause macrophage M2-M1 polarization at the maternal-fetal interface.
Infection-induced preterm delivery involves a pro-inflammatory state, but not macrophage M2-M1 polarization, at the maternal-fetal interface (HUM8P.334)
Authors: Lehr Marcus , StLouis Derek , Arenas-Hernandez Marcia , Hassan Sonia , Romero Roberto , Gomez-Lopez Nardhy
Published: January 2023

Objective: Endotoxin treatment causes preterm delivery in mice, and this is associated with an expansion of regulatory T cells (Tregs) in primary lymphoid organs (Arenas-Hernandez et al. unpublished data). Our aim was to determine whether this expansion causes a pro-inflammatory state and/or macrophage M2-M1 polarization at the maternal-fetal interface. Methods: Pregnant C57BL/6 mice were injected with either 15 μg of LPS (E. coli 0111:B4) or 200 μl PBS as a control (n=9-11 each). Mice were euthanized just prior to preterm delivery. Uterine and decidual cell suspensions were prepared and analyzed by flow cytometry to identify CD4+ and CD8+ Tregs, Th9 cells, Th17 cells, T-bet+ T cells, neutrophils, macrophages, IL4- and IFNγ-producing T cells, and M1 and M2 macrophages. Results: 1) Uterine, but not decidual, CD4+ Tregs were fewer in LPS treated mice than in controls (p=0.019); 2) Uterine and decidual macrophages were fewer in LPS treated mice than in controls (p=0.025 and 0.036); 3) Uterine and decidual neutrophils were greater in LPS treated mice than in controls (p=0.002 and 0.0001); 4) Decidual IFNγ-producing T cells were fewer in LPS treated mice than in controls (p=0.036); 5) M1 and M2 macrophage populations did not change with LPS treatment. Conclusions: Endotoxin treatment of pregnant mice promotes a pro-inflammatory stage, which includes abundant neutrophils and scarce regulatory T cells and macrophages (M1 and M2) at the maternal-fetal interface.
Replenishment of granuloma macrophages promotes mycobacterial resistance by preventing extracellular bacterial growth (INC7P.410)
Authors: Pagan Antonio , Yang Chao-Tsung , Swaim Laura , Ramakrishnan Lalita
Published: January 2023

Pathogenic mycobacteria exploit the early tuberculous granuloma for their expansion by inducing infected macrophage apoptosis and accelerating uninfected macrophage recruitment and infection upon engulfing the dying macrophages. Whether sustained macrophage recruitment to established granulomas is also host-detrimental is unclear. We addressed this question in the zebrafish model of tuberculosis by manipulating the macrophage colony stimulation factor 1 (CSF-1) pathway, which promotes macrophage development. Early granuloma formation was normal in CSF-1 receptor-deficient fish in spite of having fewer macrophages. However, they became hypersusceptible upon depleting their macrophages, leading to the sudden loss of new recruits that would normally engulf the infected dead cells. The unphagocytosed infected macrophages underwent necrosis, releasing mycobacteria into the extracellular milieu that promotes exuberant growth. Partial macrophage depletion with lipo-clodranate produced the same effects in wild-type fish. Conversely, overexpression of CSF-1 in wild-type fish increased macrophage numbers and reduced susceptibility to infection. These results suggest that granuloma kinetics must be nuanced to promote resistance - while delaying granuloma formation is host-beneficial, abolishing macrophage recruitment to established granulomas is detrimental. These results might also explain why monocytopenias confer susceptibility to mycobacterial infections in humans.
Effect of M2bMφ elimination on the resistance of alcoholic mice to infections with Klebsiella pneumoniae and Enterococcus faecalis (INC7P.414)
Authors: Cannon Krystal , Ito Ichiaki , Asai Akira , Suzuki Sumihiro , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Alcoholic patients are known to be susceptible to various opportunistic infections. Previously, we have described how mice with chronic alcohol consumption (alcoholic mice) are susceptible to opportunistic infections. IL-10+CCL2+ neutrophils (PMN-II) and IL-10+CCL1+ macrophages (M2bMφ) appearing in association with alcohol consumption were characterized as the responsible cells for impaired host antibacterial innate immunities. In this study, we tried to improve the host antibacterial resistance of alcoholic mice through the control of alcohol consumption-associated M2bMφ. Since an important role of CCL2 on the appearance of M2bMφ has been described, two inhibitors (CCL2 antisense ODN, 10 μg/mouse; glycyrrhizin, 10 mg/kg, Minophagen Pharmaceutical Co., Ltd.) for CCL2 production were utilized as controllers of M2bMφ. Alcoholic mice (oral gavage of 0.1 g/day ethanol for 10 to 12 weeks) were infected with 106 CFU/mouse of E. faecalis orally or 103 CFU/mouse of K. pneumoniae intratracheally. In the results, severe bacterial growth was seen in the liver and spleen of alcoholic mice exposed to E. faecalis and K. pneumoniae. However, bacterial growth was greatly inhibited in the organs of the mice treated with CCL2 antisense ODN or glycyrrhizin. M2bMφ generation was also inhibited in both groups of treated mice. These results indicate that host resistance against E. faecalis and K. pneumoniae infections can be improved in alcoholic mice after the depletion of M2bMφ.
Bactericidal IL-12+ Mφ generation in sub-acutely burned mice infected with MRSA after treatment with CCL1 antisense oligodeoxynucleotides (ODN) (INC7P.415)
Authors: Nishiguchi Tomoki , Nakamura Kiwamu , Herndon David , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Sub-acutely burned mice are shown to be carriers of M2bMφ, these cells are characterized as inhibitor cells for Mφ conversion from resident Mφ to M1Mφ. CCL1 released from M2bMφ has been identified as an essential chemokine for the maintenance of M2bMφ properties, and M2bMφ derived from sub-acutely burned mice have reverted to resident Mφ after cultivation with CCL1 antisense ODN. In this study, we tried to determine the effect of CCL1 antisense ODN on M1Mφ generation in sub-acutely burned mice infected with MRSA. Two weeks after burn injury, the mice were infected i.v. with 105 CFU/mouse of MRSA, and then, treated with the ODN (s.c., twice a day, 10 μg/mouse). Two days after the infection, F4/80+ cells were isolated from the peritoneal cavities of the mice and assayed for IL-12+ cells. Also, the killing activity of these cells against MRSA was tested in vitro. In the results, a majority of Mφ from normal mice infected with MRSA were shown to be IL-12+ cells (>82%), while 10% or less of Mφ from burn mice infected with the pathogen were IL-12+ cells. However, a majority of Mφ isolated from burn mice infected with MRSA and treated with the ODN were shown to be IL-12+ cells. A majority of Mφ from burn mice infected with MRSA were not bactericidal, while Mφ preparations from the same mice additionally treated with the ODN were bactericidal. These results indicate that M1Mφ are induced after treatment with CCL1 antisense ODN in sub-acutely burned mice infected with MRSA.
Critical involvement of GPCR kinase-5 in E.coli induced pneumonia (INC7P.427)
Authors: Packiriswamy Nandakumar , Steury Michael , Parameswaran Narayanan
Published: January 2023

G-protein coupled receptor kinases are serine/threonine kinases involved in a range of pathophysiological processes including inflammation. We recently demonstrated, deficiency of G-protein coupled receptor kinase-5 (GRK5) improves sepsis outcome in a polymicrobial septic. In this study we examined the role of GRK5 in various immunopathological features associated with E.coli induced pulmonary infection using wild type (WT) and GRK5 knockout (KO) mice. Interestingly, 12-hours post-infection, GRK5KO exhibited significantly reduced inflammation and bacterial load as well as decreased neutrophils in the lung. Ex vivo assays demonstrated, GRK5KO and WT neutrophils have similar capacity to kill bacteria suggesting an early increase in neutrophils in the KO mice might decrease bacterial load. Consistent with this prediction, 4 hours post-infection, GRK5KO mice had markedly elevated neutrophil numbers in the lungs, compared to the WT mice. Interestingly, at later time points after infection (12 hours) even though the neutrophil (Ly6G+) numbers were reduced in the KO, the number of efferocytosing macrophages (Ly6g+ F4/80+) were markedly higher in the GRK5KO animals, suggesting that GRK5 deficiency advances the inflammatory resolution phase (increased efferocytosing macrophages). Taken together, our results show that GRK5 deficiency improves pulmonary infection and inflammation likely via early-enhanced neutrophil recruitment as well as swifter clearance of neutrophils in the lung.
Low grade endotoxemia delays wound healing associated with inflammatory monocytes/macrophages expansion (INC7P.405)
Authors: Geng Shuo , Chen Keqiang , Yuan Ruoxi , Li Liwu
Published: January 2023

Low grade endotoxemia, mainly caused by subclinical level of blood circulating LPS, is prevalent in the individuals with adverse health conditions or lifestyles. Here, we found that low doses LPS (50 pg/ml~1 ng/ml) preferentially expanded Ly6C+ monocyte population in bone marrow cultures supplemented with M-CSF. TGF-β production in monocytes was diminished by low dosed LPS. Interestingly, high doses LPS (10 ng/ml~1 µg/ml) decreased Ly6C+ monocyte population. Low doses LPS dramatically enhanced IL-12 production but reduced iNOS expression in macrophages, while high doses LPS exerted an opposite impact. In a mouse model with wound punctures on the back skin, super low grade LPS administration (5 µg/kg) remarkably delayed wound repair, as shown by three days’ lag in wound closure compared to PBS injected mice. We observed a two-fold reduction of collagen deposition around wound sites, suggesting disruption of tissue remodeling. Consistent with in vitro studies, we detected increased numbers of Ly6C+ monocytes circulating in peripheral blood and accumulated in wound lesions in the mice with LPS treatment. Moreover, TGF-β production in the skin, which facilitates wound repairing, was also remarkably impaired by low grade LPS administration. These data demonstrated that subclinical level of LPS was sufficient to prime monocytes/macrophages and compromise wound healing process, potentially due to the expansion of monocytes/macrophages with low-grade pro-inflammatory properties.