bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel orally active ligands of lanthionine synthetase C-like protein 2 ameliorate influenza-related inflammation and immunopathology (IRM7P.482)
Authors: Philipson Casandra , Lu Pinyi , Bassaganya-Riera Josep , Vento Salvador , Carbo Adria , Viladomiu Monica , Kale Shiv , Zhang Xiaoying , Uren Aykut , Kallarakal Abraham , Xu Bin , Hontecillas Raquel
Published: January 2023

The use of broad-based drugs that target the host inflammatory response and facilitate recovery has been proposed for treating influenza. We previously identified lanthionine synthetase C-like protein 2 (LANCL2) as a new therapeutic target for inflammatory diseases, predicted in silico that NSC61610 and abscisic acid (ABA) bind to LANCL2 and demonstrated their therapeutic efficacy in mouse models of colitis. Here, we use surface plasmon resonance to demonstrate for the first time that NSC61610 binds to LANCL2, promotes cAMP accumulation and protein kinase A (PKA) phosphorylation in RAW macrophages. To examine the therapeutic efficacy of NSC61610 in vivo, we infected mice with influenza A H1N1pdm virus and determined its impact on influenza-related morbidity, mortality, lung inflammatory profiles and histopathology. Our results demonstrate that oral administration of NSC61610 (20 mg/kg/day) ameliorates influenza by down-modulating pulmonary TNF-α and MCP-1 mRNA expression and reducing the infiltration of neutrophils and pro-inflammatory monocytes in the lungs. The therapeutic actions and signaling of NSC61610 were abrogated following gene silencing by LANCL2 siRNA administration further validating our findings. Thus, binding of NSC61610 to LANCL2 and signaling through cAMP and PKA represents a novel mechanism for modulating pulmonary host responses and improving disease resolution in influenza virus-infected mice.
The role of GCN2 in type-2 mucosal inflammation within the lung (IRM7P.483)
Authors: Bradley Jillian , Liu Haiyun , Ravishankar Buvana , Huang Lei , Perdue Aja , Urban Joseph , McGaha Tracy
Published: January 2023

Infection with the parasitic roundworm Nippostrongylus brasiliensis causes lung damage associated with an inflammatory response dominated by alternative (type-2) inflammation. Macrophages are requisite mediators of lung inflammation and pathology resolution in the N. brasiliensis infection model. Metabolic stress resulting from enzymatic consumption of amino acids engages the GCN2 arm of the integrated stress response, which profoundly alters the inflammatory potential of antigen presenting cells, causing them to suppress type-1 cytokine expression and promote type-2 cytokine expression. Thus, we hypothesized that GCN2 activation, as a result of infection driven nutrient stress, may be a key mechanism promoting M2 macrophage differentiation and type-2 inflammatory processes. When we examined cytokine expression in the lungs of GCN2KO mice infected with N. brasiliensis, we found significantly decreased levels of IL-10 relative to WT controls. This was associated with increased lung infiltration of macrophages that expressed reduced expression of FIZZ1 and YM-1, indicative of a skewing towards an M1 phenotype. Moreover, we observed dramatic increases in lung pathology associated with increased cellular infiltrate and fibrosis in infected GCN2KO mice suggestive of severe helminth-driven lung inflammation. Thus, our findings suggest a novel regulatory role for GCN2 kinase during type-2 inflammatory processes in the lung.
ATAD5 deficiency decreases B cell division and Igh recombination (IRM8P.700)
Authors: Zanotti Kimberly , Gearhart Patricia
Published: January 2023

Mammalian ATAD5 and its yeast homolog ELG1 are responsible for unloading PCNA from newly synthesized DNA. Prior work in HeLa and yeast cells showed that a decrease in ATAD5 protein levels resulted in accumulation of chromatin-bound PCNA and slower cell division. We used B cells from heterozygous (Atad5+/m) mice to examine the effects of PCNA buildup and decreased cell proliferation on antibody diversity. ATAD5 haploinsufficiency did not affect the frequency or spectrum of somatic hypermutation in antibody genes from Peyer’s patch B cells, indicating that PCNA accumulation did not alter DNA repair or DNA polymerase η usage at the Igh locus. However, following ex vivo stimulation, Atad5+/m B cells were delayed during S-phase cell cycle progression and had reduced cell proliferation compared to wild type cells. These cells had a significant decline in activation-induced deaminase expression, resulting in decreased double-strand breaks in the switch region and inter-chromosomal translocations. Class switch recombination was also reduced, although the types of end-joining pathways were not affected. Our results describe a defect in DNA replication that directly affects Igh recombination via reduced cell division.
B cell-intrinsic mTOR signaling regulates the generation of germinal center B cells (IRM8P.716)
Authors: Jones Derek , Chernova Irene , Allman David
Published: January 2023

Germinal centers (GCs) are sites enriched for proliferative B cells undergoing class switch, somatic hypermutation, and selection for clones expressing high-affinity antigen-specific receptors. GCs are a main source of long-lived plasma cells (PCs) and memory B cells, and many B-lineage cancers including multiple myeloma (MM), a malignancy of PCs, also originate in GCs. However, little is known about the biochemical pathways regulating GC B cells or GC-derived effectors. Thus, we sought to define the role of mammalian target of rapamycin (mTOR), a serine/threonine kinase known to regulate several biochemical pathways needed for cell growth, division, and survival, in the maintenance of GC B cells and GC-derived cells. To address the role of mTOR in GC B cell generation, we treated NP-CGG-immunized mice with rapamycin, an mTOR inhibitor. Rapamycin treatment caused a near-complete loss of NP-specific GC B cells in the spleen, and a selective reduction of newly formed bone marrow PCs. We next generated Raptorf/f x CD20-ER-Cre mice to induce B cell-specific deletion of the mTOR-requisite protein Raptor; Raptor deletion caused the ablation of antigen-specific GCs, as well as secondary antibody responses mediated by memory B cells. The data indicate that GC B cells utilize mTOR signaling to regulate metabolic pathways required for cell division and/or survival. Future studies will address the notion that malignant PCs responsible for MM must be replenished by ongoing GC responses.
Immunoglobulin isotype switching during early B cell development (IRM8P.702)
Authors: Kumar Satyendra , Kenter Amy
Published: January 2023

During B cell lymphopoiesis in the bone marrow (BM), V(D)J joining assembles immunoglobulin (Ig) VH and VL genes mediated by RAG recombinase. Mature splenic B cells undergo class switch recombination (CSR) and somatic hypermutation initiated by activation induced deaminase (AID). However, the strict ontological separation of these processes may breakdown under some circumstances. Several immunodeficiencies including Omenn syndrome display increased serum IgE despite undetectable numbers of B cells. We hypothesized that CSR occurs in BM pro- or pre-B cells. Earlier, we showed that Rag1 and Mb1 deficient pro-B cells can be induced to undergo robust CSR, albeit to a restricted subset of IgH isotypes, including IgG2b and IgE. Immunization of Rag1-/- mice with LPS, promoted IgG2b switching in BM pro-B cells. We demonstrated that V(D)J joining can be induced to follow CSR. We found a unique stage specific chromatin conformation of Igh locus in pro-B cells that may repress IgG3 and IgG1 switching (Genes Dev. 27:2439). Here we show that CSR is inducible in WT pro-B cells. Using an elegant mouse model to irreversibly mark AID expressing cells, a knockin carrying AIDcre/+RosaYFP (Nat Imm. 10:1292), we assess the frequency of AID expression in BM pro-B cells activated in vitro and in vivo with LPS. These findings have implications for the hyper-IgE phenotype in Omenn syndrome, autoimmune repertoire development and leukemagenesis via the co-expression of the RAG and AID recombinases.
Assessing B lymphocyte clonal diversity and expansion by high-throughput sequencing of rearranged IgH loci from naïve and memory repertoires (IRM8P.708)
Authors: Emerson Ryan , DeWitt William , Lindau Paul , Sherwood Anna , Williamson David , Rieder Mark , Chung Moon-Wook , Carlson Christopher , Robins Harlan
Published: January 2023

Diversity in B cell antigen specificity arises from structural diversity of the B cell receptor generated by somatic rearrangement of the immunoglobulin heavy (IgH) and light chain loci during B cell development. Antigen-experienced (memory) B cells are stimulated to proliferate upon successful binding of antigen, whereas the extent to which antigen-inexperienced (naïve) cells proliferate, and therefore the total diversity of circulating naïve B cells, is unclear. High-throughput sequencing of rearranged IgH CDR3 regions was performed for sorted memory and naïve B cell samples from each of 3 adults. For each sample, approximately 5 million B cells were split among 188 libraries for multiplex PCR amplification of IgH CDR3 segments, allowing us to estimate clone abundances using the number of libraries occupied by each distinct CDR3 segment. Modeling sample clone abundances as a superposition of homogeneous Poisson processes, memory repertoire clone abundance was characterized. Data from naïve samples suggest extreme diversity with more than 98% of clones occupying only one library (i.e., almost surely present in only one cell in the sample). A measure-theoretic upper bound on mean clone abundance in the naive repertoire was computed based on Monte Carlo simulation, indicating that naïve B cells typically undergo little, if any, expansion. We estimate the diversity of circulating naive B cells to be on the order of 100 million in healthy adults.
Immunohistochemical and multicolor flow cytometric analysis of STAT1 (pY701) expression in human and rat cells (IRM9P.733)
Authors: Ernst David , Ji Xiang Dong , Gao Guo-Jian , Wang Xiao , Berry Jody
Published: January 2023

Signal Transducer and Activator of Transcription 1 (STAT1) is phosphorylated at tyrosine 701 (pY701) upon cellular activation by interferons. This site-specific phosphorylation activates the signaling activity of STAT1 that translocates to the nucleus and regulates target gene transcription, which determines cellular growth, proliferation, and effector functions. An antibody that specifically recognizes STAT1 (pY701) was developed to study STAT1 roles played in experimental systems. Human tonsil sections, briefly cultured with a phosphatase inhibitor, revealed enhanced immunohistochemical (IHC) staining of nuclear STAT1 (pY701) in lymphocytes and epithelial cells when compared with untreated or phosphatase-treated controls. Similarly, in an in vivo model, liver sections prepared from rats treated with a phosphatase inhibitor revealed enhanced IHC staining of nuclear STAT1 (pY701) in hepatocytes compared with controls. The Ab also served as a specific probe in multicolor flow cytometric analyses designed to characterize the nature of lymphoid cells that express STAT1 (pY701) in response to cytokines. Exogenous interferon-alpha treated CD4+ and CD8+ T cells, B cells, monocytes and NK cells all were induced to express STAT1 (pY701) as determined by multicolor flow cytometric analysis. The applications of this Ab, as well as additional Abs specific for phosphorylated STATs 3-6, will be presented and discussed in terms of their use for cell analysis across multiple applications.
SAP facilitates recruitment and activation of LCK at NTB-A receptors during restimulation-induced cell death. (IRM10P.734)
Authors: Katz Gil , Krummey Scott , Larsen Sasha , Stinson Jeffrey , Snow Andrew
Published: January 2023

Upon TCR restimulation, activated, cycling T cells can undergo a self-regulatory form of apoptosis known as restimulation-induced cell death (RICD). We previously demonstrated that RICD is impaired in T cells from patients with X-linked lymphoproliferative disease (XLP), which lack SLAM-associated protein (SAP) expression. Both SAP and the specific SLAM receptor NK, T, and B cell antigen (NTB-A) are required for RICD, but the mechanism by which these molecules promote a strong, pro-apoptotic signal through the TCR remains unclear. Here we show that the Src-family kinase LCK, but not FYN, associates with NTB-A in activated human T cells. This association increased after TCR restimulation in a SAP-dependent manner, requiring both immunoreceptor tyrosine-based switch motifs (ITSMs) in the NTB-A cytoplasmic tail. Both NTB-A-associated LCK phosphorylation and kinase activity were enhanced in restimulated T cells, amplifying proximal TCR signaling. In contrast, TCR-induced LCK association with NTB-A, as well as phosphorylation and kinase activity, were reduced in XLP patient T cells or normal T cells transfected with SAP-specific siRNA, consistent with RICD resistance. Collectively, our data reveal how SAP nucleates a previously unknown signaling complex involving NTB-A and LCK to potentiate RICD of activated human T cells.
Analysis of human TCRβ repertoire by a RARE-sequencing method reveals distinct features of TCRβ diversity and distribution and their alterations with age (IRM10P.758)
Authors: Weng Nan-ping , Shi Alvin , Li Hoi-Ming , Hiroi Toyoko , Zhang Yongqing , Chen Guobing , Metter Jeffrey , Ferrucci Luigi
Published: January 2023

The T cell receptor (TCR) repertoire (diversity and distribution) is a vital parameter of the overall function of T cells. Reduction of TCR diversity and alteration of TCR distribution is believed to occur with age and acts as a major contributor for age-associated decline of immune function. However, neither the actual size of the TCR repertoire nor its precise age-related change have been directly determined. Applying a RACE-next generation sequencing (RAP-Seq) method, we assessed the TCRβ repertoire of blood CD4+ and CD8+ T cells of 13 adults (21-94 years old) including 6 adults with longitudinal samples. We found that TCRβ diversity of CD4+ T cells is 3-4 times greater than CD8+ T cells. TCRβ CDR3 was distinct between CD4+ and CD8+ T cells with preferential amino acid usage. Reduced TCRβ diversity and expanded highly abundant TCRβ clones occurred from middle to old subjects (45-75 years old) but not in very old subjects (80-94 years old). Together, these findings reveal the distinct features and actual age-associated changes of TCRβ repertoire, which could serve as a measure of the immune competency and a guide for the clinical intervention in older humans.
Co-expression of CD40 and EBV-associated latent membrane protein (LMP)1 results in altered downstream signaling responses in B cells (IRM11P.762)
Authors: Boody Christopher , La Porta James , Valentin-Acevedo Anibal , Covey Lori
Published: January 2023

The binding of CD40 on B cells to CD154 on activated CD4 T cells is essential for regulating humoral responses in antigen-primed B cells. The EBV transforming protein, LMP1 mimics many aspects of CD40 function through the binding of an overlapping set of TNF receptor-associated factor (TRAF) signaling molecules. Co-expression of LMP1 and CD40 occurs at distinct stages of EBV infection and in tumor cells in which EBV is an etiological agent. Although both molecules share functional homology, the signaling cross talk between LMP1 and CD40 is not well understood. Here we show that LMP1 directly increases both TRAF3 and CD40 in B cells through elevated RNA expression. Additionally, LMP1 enhances the association of CD40 and TRAF3 with lipid rafts. To better understand LMP1-specific changes in CD40 signaling we evaluated the activity of specific downstream targets in response to CD154 stimulation using phospho-specific flow cytometry. Stimulated B cells produced a greater signal for p38 and p65 than those constitutively expressing LMP1. Also, CD40 signaling reduced the activity of LMP1-induced pLCγ2 whereas LMP1 significantly lowered that of Akt in cells receiving both stimuli. Together, our results demonstrate that LMP1 increases expression of CD40 and TRAF3 and specifically modifies CD40 activation responses when both proteins are concurrently active. These changes have the potential of underlying differences in B cell activation, survival and differentiation.
Inhibition of IL-6 signaling by zinc leads to repression of the Th17 response (IRM11P.763)
Authors: Lee Won-Woo , Lee Hyunju , Yoon BoRuem , Hwang Yuri
Published: January 2023

Zinc is an essential trace element that plays pivotal roles in multiple facets of the immune system. Besides its catalytic and structural roles, zinc also functions as an intracellular signaling molecule, and changes in zinc levels can cause both direct and indirect modulation of immune responses. Further, cytoplasmic levels of bioavailable zinc in immune cells are largely influenced by many extracellular stimuli. Here we provide evidence that zinc alters the cytokine production profile of human CD4 T cells by functioning as an intracellular signaling molecule during T-cell responses. In vitro zinc treatment of T cells in the presence of activated monocytes inhibited IFN-γ and IL-17 producing cells, but not IL-4 producing cells. Of note, production of IL-17+ CD4 T cells, which is significantly upregulated by LPS-stimulated monocytes, was preferentially repressed by zinc. Increased cytoplasmic zinc in T cells suppressed IL-6 signaling via repression of phosphorylation of Stat3, thus leading to an inhibitory effect on Th17 responses facilitated by monocyte-derived IL-6 in humans. These finding suggest that therapeutic manipulation of zinc bioavailability may be a good means by which to modulate CD4 T-cell responses.
The origin of natural antibody (LYM6P.764)
Authors: Reynolds Alexander , Kuraoka Masayuki , Kelsoe Garnett
Published: January 2023

Natural antibody is constitutively present in the serum of all mammals. This unique IgM plays an important role in the clearance of cellular debris, the early control of viral and bacterial expansion, and the initiation of T-dependent humoral responses. Despite decades of study, the origins of natural antibody have not been precisely defined. Transfer experiments have shown a close correlation between peritoneal cells, including B1a cells, and natural antibody, but peritoneal B1 cells do not secrete adequate amounts of IgM to support the levels found in mouse serum. Here, we show that a discrete population of IgM plasmacytes in the bone marrow produces all serum IgM in resting mice. These plasmacytes originate from cells in the peritoneal cavity, but not from B1a B cells, and transfer of these progenitors completely restores serum IgM and the specific compartment of bone marrow plasmacytes in Rag1 deficient mice. This plasmacyte compartment contains a substantial fraction of non-dividing, long lived cells that do not occupy the IgG plasmacyte survival niche in the bone marrow but instead depend on IL-5. Genetic analysis of these IgM plasmacytes reveals an unmutated, clonally diverse population strongly enriched for VH11 rearrangements. We propose that these natural IgM plasmacytes represent an “innate B-cell” population descended from yolk sac progenitors, and that natural IgM plasmacytes are not the progeny of B1 B cells but instead share an earlier common ancestor.
Each naïve B cell within a polyclonal repertoire produces a different frequency and pattern of effector cells (LYM6P.769)
Authors: Taylor Justin , Pape Kathryn , Jenkins Marc
Published: January 2023

Following activation with antigen, naïve B cells undergo a robust proliferative expansion and differentiation that results in the production of numerous distinct cell types with different roles in protective immunity. In fact, germinal center B cells, memory B cells, plasma cells and an undifferentiated population of T/B border B cells are all detected 7 days following immunization. While some of the transcriptional regulators and extrinsic factors directing differentiation of these subtypes are known, these mechanisms cannot account for the simultaneous existence of all 4 cell subtypes. By tracking the response of a single naïve B cell within a polyclonal response we find that an individual naïve B cell usually contributes half of the subtypes found in a polyclonal response. In fact, the progeny of only 8% of cells analyzed contained all 4 subtypes while 12% of cells only generated a single subtype. One factor constraining differentiation is B cell receptor affinity for antigen, but individual cells with identical receptors do produce different patterns of progeny. Thus, the heterogeneity found within the polyclonal response is in part the result of the differential responses of individual naïve B cells.
Lig4 modulates p53-p21 checkpoints and inhibits A-EJ during end-joining repair phase of IgH class switching (LYM6P.770)
Authors: Park Jihye , Welner Robert , Tenen Daniel , Yan Catherine
Published: January 2023

Mutations in the non-homologous end-joining DNA repair protein DNA Ligase IV (LIG4) causes a human syndrome of immunodeficiency and radiosensitivity. We previously described knock-in mice with a homozygous R278H mutation in Lig4 (Lig4R/R) that displayed severely leaky B lymphocyte development, resulting in near lack of mature B cells yet only a partial block in IgH class switch recombination (CSR). Here we show that diminished DNA ligase 4 activity, resulting in slower kinetics of DNA double strand break (DSB) repair and attenuation of p53-dependent DNA double strand break (DSB) response, to underlie the partial CSR block in the Lig4R/R activated B cells. Analysis of p53 levels in activated Lig4R/R B cells revealed increased proteosome mediated p53 decay and p21 activation, indicative of slower repair kinetics, effects mimicked in the human Lig4 R278H 180BR cell line. Concurrent p53 inactivation had little effect on the frequency of IgH breaks; but substantially slowed the cell cycle progression of activated Lig4R/Rp53-/- B cells, and failed to promote the development of B-lymphomas that recurrently developed in murine Lig4-/-p53-/- settings. Analysis Lig4R/R CSR junctions also revealed the predominance of NHEJ instead of NHEJ-independent A-EJ in their generation. Hence NHEJ maintains genomic stability by modulating the p53 DNA damage response and inhibiting A-EJ. These findings provide an explanation for the complex B-lymphoid phenotypes of the human Lig4 Syndrome.
The Pseudomonas aeruginosa virulence factor Cif inhibits the generation of the pro-resolving lipid mediator 15-epi lipoxin A4 (MPF3P.812)
Authors: Flitter Rebecca , Ono Emiko , Hvorecny Kelli , Levy Bruce , Madden Dean , Bomberger Jennifer
Published: January 2023

Pseudomonas aeruginosa is an opportunistic Gram-negative bacteria that causes a wide range of severe nosocomial infections, especially in immunocompromised patients with chronic inflammatory lung disease. P. aeruginosa infections are characterized by persistent and robust neutrophilic inflammation which can cause damage to host tissue. Lipoxins, including 15-epi-lipoxin A4 (15-epi-LXA4), are lipid mediators that play a critical role in resolving inflammation and promoting tissue homeostasis. The production of 14,15-epoxy-eicosatrienoic acid (14,15-EET) by airway epithelial cells stimulates the generation of 15-epi-LXA4 by neighboring neutrophils through transcellular biosynthesis. We have identified a P. aeruginosa virulence factor with epoxide hydrolase activity called cystic fibrosis inhibitory factor (Cif), which we hypothesize disrupts the generation of 15-epi-LXA4. We show that both P. aeruginosa and recombinant Cif protein alone hydrolyzes the epoxide moiety of (14,15-EET), whereas a Cif mutant P. aeruginosa or catalytically dead D129S mutant of Cif have no effect. Furthermore, the Cif-mediated reduction of 14,15-EET inhibited the generation of 15-epi-LXA4 by activated neutrophils in vitro. The D129S mutant Cif was also incapable of reducing 15-epi-LXA4 biosynthesis. Taken together, our data suggest a novel model whereby the P. aeruginosa virulence factor Cif reduces 15-epi-LXA4 in the airways, thus promoting persistent inflammation and consequent lung damage.
NanoCurcumin inhibits TNF-α in Mycobacterium tuberculosis-infected macrophages (MPF3P.821)
Authors: Pokkali Supriya , Ordonez Alvaro , Chenna Venugopal , Maitra Anirban , Jain Sanjay
Published: January 2023

Background: Host response to M. tuberculosis (M.tb) infection leads to tissue destruction and impairs the ability of drugs to reach the bacteria. New anti-inflammatory adjuvant therapies could enhance bacterial clearance. Curcumin (diferuloylmethane) is a lipophilic phytochemical present in turmeric (Curcuma longa). Encapsulation of curcumin with polymers (NanoCurcuminTM) increased absorption and bioavailability of curcumin and is being investigated as host-directed therapies for the treatment various diseases. Methods and Results: We investigated the effect of free and encapsulated NanocurcuminTM on M.tb induced host inflammatory responses in M.tb infected J774 macrophages. Bacterial burden and cytokine levels were enumerated by standard microbiological and immunological methods. We utilized physiologically achievable levels of NanocurcuminTM (based on prior in vivo studies) and found a dose dependent decrease in the levels of TNF-α in M.tb infected J774 macrophages. Conclusions: NanoCurcumin™ can modulate TNF-α and could be utilized as a host-directed therapy to enhanced bacterial clearance and potentially shorten TB drug regimen.
The role of IL-23 in regulating the function of phagocytic cells during Listeria monocytogenes infection (MPF3P.822)
Authors: Indramohan Mohanalaxmi , Break Timothy , Witter Alexandra , Berg Rance
Published: January 2023

The IL-23/IL-17 axis contributes to autoimmune disorders, but is protective against certain pathogens, particularly extracellular bacteria. We have previously shown that IL-23 provides resistance against the intracellular bacterium, Listeria monocytogenes (LM) by mediating the recruitment of neutrophils to the liver, and inflammatory monocytes to the spleen. However, it is not known whether IL-23 can impact the function of phagocytic cells including inflammatory monocytes, neutrophils, and macrophages, during LM infection. Phagocytic cells express receptors for IL-23 and IL-17A suggesting the activity of these cells could be regulated by IL-23 or IL-17A. Surprisingly, phagocytic cells from mice lacking IL-23 (IL-23p19 knockout) had equivalent phagocytic potential and ROS production compared to C57BL/6 mice. Additionally, exogenous stimulation with rIL-23 or rIL-17A did not induce or enhance the production of pro-inflammatory mediators from splenocytes isolated from C57BL/6 mice. Therefore, IL-23 does not impact the function of phagocytic cells either by a direct or an indirect mechanism during LM infection. Collectively, our data reveal that the enhanced susceptibility of IL-23p19 knockout mice is not due to functional impairment of phagocytic cells; instead it is caused by the inefficient recruitment of neutrophils to the liver, and inflammatory monocytes to the spleen, resulting in a reduction in the overall levels of TNF-α and nitric oxide during LM infection.
Taenia crassiceps excreted/secreted products induce alternatively activated macrophages in vitro. (MPF3P.824)
Authors: Martínez-Saucedo Diana , Faz Berenice , Callejas Blanca , Ledesma-Soto Yadira , Terrazas Luis I.
Published: January 2023

Alternatively activated macrophages (aaMφ) are key players in the immunomodulation generated during helminth infections. Recent evidences suggest that derived molecules from helminths can directly induce aaMφ. Taenia crassiceps and its excreted/secreted products (TcES) have been demonstrated in modulate inflammatory responses. We have special interest in determine whether TcES directly are capable of generate aaMφ. On the other side, the ability of microRNAs (miRNAs) to influence cellular functions and their development have been suggested as a mechanism to develop new therapeutic applications based on differential expression of miRNAs. Because of this, is quite important to characterize the miRNAs involved in the response of Mφ to TcES as a possible mechanism to control inflammation as well as contributing to a better understanding of the mechanisms involved in macrophage polarization. We have found that macrophages derived from bone marrow (MφBM) exposed directly to TcES expressed molecules related to alternative activation such as PD-L1 and Mannose receptor, as well an downregulation in miR-155 and let-7i. Our data suggest that TcES induce aaMφ where some miRNA induction may participate.
The adaptor CARD9 is required for adaptive but not innate immunity to oral mucosal Candida albicans infections (MPF3P.805)
Authors: Bishu Shrinivas , Hernandez-Santos Nydiaris , Simpson-Abelson Michelle , Huppler Anna , Conti Heather , Ghilardi Nico , Mamo Anna , Lin Xin , Gaffen Sarah
Published: January 2023

Oropharyngeal candidiasis (OPC, thrush) is an opportunistic infection caused by the commensal fungus Candida albicans. OPC is common in individuals with HIV/AIDS, infants, and individuals with congenital immune defects. Immunity to OPC is dependent on the IL-23/IL-17R axis, as mice and humans with defects in IL-17R signaling (IL17F, ACT1, IL-17RA) or in genes that direct Th17 differentiation (STAT3, STAT1, CARD9) are prone to mucocutaneous candidiasis. Conventional Th17 cells are induced in response to C. albicans via signals from C-type lectin receptors, which signal through the adaptor CARD9 leading to production of Th17-inducing cytokines such as IL-6, IL-1 and IL-23. Recent data indicates that IL-17 can also be made by numerous innate cell subsets. These innate ‘Type 17’ cells resemble conventional Th17 cells, but they can be activated without need for prior antigen exposure. Because C. albicans is not a commensal organism in rodents and thus mice are naïve to this fungus, we had the opportunity to assess the role of CARD9 in innate versus adaptive responses using an OPC infection model. As expected, CARD9-/- mice failed to mount an adaptive Th17 response following oral Candida infection. Surprisingly, however, CARD9-/- mice had preserved innate IL-17-dependent responses and were almost fully resistant to OPC. Thus, CARD9 is important primarily for adaptive immunity to C. albicans, whereas alternate recognition systems appear to be needed for effective innate responses.
Lung hydrolases modify the course of Mycobacterium tuberculosis infection in vivo (MPF3P.806)
Authors: Moliva Juan , Sasindran Smitha , Arcos Jésus , Turner Joanne , Torrelles Jordi
Published: January 2023

Mycobacterium tuberculosis (M.tb) kills one person every 18 seconds. Upon M.tb deposition in the airways, bacilli are bathed in alveolar lining fluid (ALF). Our published data indicate that hydrolytic enzymes present in ALF modify the cell wall of M.tb with two outcomes: cell wall modifications and release of cell wall fragments into the milieu. These modifications resulted in an altered macrophage immune response in vitro. Here we used the low dose aerosol mouse model of M.tb infection to assess if ALF/hydrolase-derived modifications of M.tb alter the immune response to infection in vivo. Our current data indicate that ALF/hydrolase-derived modifications on the M.tb cell wall led to increased CD4 and CD8 T cell numbers and activation in the lung, and altered cytokine secretion (increased TNF, IL-12 and IFN-γ), important for control of M.tb infection. These results correlated with an improvement of the M.tb burden within the lung starting on day 21 of infection. Thus, our results support the concept of human ALF hydrolases being novel immune components that can influence the generation of immune responses during M.tb infection.
Symbiotic bacterial metabolites regulate GI barrier function via PXR and TLR4 (MUC4P.852)
Authors: Khanna Kamal , Mukherjee Subhajit , Venkatesh Madhukumar , Qiu Zhijuan , Mani Sridhar
Published: January 2023

Intestinal microbial metabolites affect mucosal integrity through incompletely characterized mechanisms. Here we identify microbial specific indoles that regulate intestinal barrier function through the xenobiotic sensor, pregnane X receptor (PXR). Indole 3-propionic acid (IPA), in the context of indole, serves as a ligand for PXR and down-regulates enterocyte TNF-α while up-regulating junctional protein-coding mRNAs. Pxr-/- mice exhibit a distinctly “leaky” gut physiology coupled with up-regulation of the Toll-like receptor (TLR) signaling pathway. These defects in the epithelial barrier are corrected in Pxr-/-/Tlr4-/- double-knockout mice. To validate that IPA drives the anti-inflammatory response directly via PXR, we exposed intestinal commensal-depleted Pxr+/+ and Pxr-/- mice to live (IPA-producing) or heat-killed (non-IPA producing) C. sporogenes preceding indomethacin exposure (toxic small intestinal injury model). There was a significant reduction in histologic injury and mucosal myeloperoxidase (MPO) enzyme activity. The intestinal mucosa exposed to live (as opposed to heat-killed) C. sporogenes had significant induction of PXR target gene (Ugt1a1). These effects were absent in Pxr-/- mice. Our results demonstrate that a direct chemical communication between the intestinal symbionts and PXR regulates mucosal integrity through a pathway which involves luminal sensing and signaling by TLR4.
Innate lymphoid cells protect host against intestinal pathogen through Id2 dependent IL-22 production and regulation of microbiota. (MUC4P.855)
Authors: Guo Xiaohuan , Kee Barbara , Fu Yang-Xin
Published: January 2023

Innate lymphoid cells (ILCs) represent a novel family of immune cells that can regulate immunity, inflammation, and tissue repair in multiple barrier surfaces. While Id2 is known to be required for the development of ILCs progenitor, Id2 remains highly expressed in differentiated ILCs with unknown function. Using newly generated conditional KO mice, we demonstrated a novel role for Id2 in RORγt+ ILC3s not only in controlling the further development and maintenance of ILC3s but also regulating IL-22 production by ILC3s through IL-23R - STAT3 pathway. Id2 deficiency in ILC3s increased morbidity and mortality after Citrobacter rodentium infection. Interestingly, the mice with Id2 deficiency in ILC3s exhibited much higher fecal C. rodentium CFU even within 24 hours post inoculation, suggesting that preexisting Id2-dependent gut microenvironment mediated the early colonization resistance against C. rodentium. Furthermore, the wild type germ free mice colonized with the microbiota from the conditional Id2 deficient mice by gavage of cecal material also had higher fecal C. rodentium CFU, compared with the germ free mice that received microbiota derived from wild type mice. Cohousing partially rescued conditional Id2 deficient mice from lethal infection. Together, our data suggest that continued Id2 expression is required for the development and function of ILC3s, and ILC3s protect host against intestinal pathogen through Id2 dependent regulation of microbiota.
Goblet cell-associated antigen passages are reduced during enteric infection (MUC4P.831)
Authors: Kulkarni Devesha , McDonald Keely , Knoop Kathryn , Miller Mark , Newberry Rodney
Published: January 2023

The intestinal mucosa is continually exposed to foreign antigens derived from the diet, commensal bacteria, and pathogens. Regulating the exposure of the immune system to this broad array of antigens is crucial to ensure intestinal homeostasis and to respond appropriately to enteric infections. Using in vivo two-photon imaging of the intestine of live mice, we recently demonstrated that under steady state conditions a subset of small intestinal goblet cells (GCs) transport luminal antigens to CD103+ dendritic cells (DCs) by forming goblet cell-associated antigen passages (GAPs). To understand how enteric infection altered GCs and GAPs, we infected mice with wildtype fluorescently labeled Salmonella typhimurium and evaluated GCs and GAPs using fluorescence microscopy. Within hours of intraluminal exposure, Salmonella preferentially localized around and within GCs that had formed GAPs. 24 hours after oral Salmonella infection, a subset of GCs were found to be apoptotic, and 72 hours after infection, GAPs were significantly decreased beyond the level of GC apoptosis. Salmonella infection resulted in epithelial activation of the epidermal growth factor receptor (EGFR), a potential regulator of GC secretion and GAP formation. These observations indicate that GAPs may provide a portal of entry for pathogens, and that GAP formation is reduced following enteric infection potentially to avoid delivery of luminal antigen to lamina propria immune cells in a hostile environment.
Trefoil factor 2 maintains and restores pulmonary function via promoting epithelial repair and M2 activation (MUC5P.859)
Authors: Herbert De'Broski , Hung Li-Yin , Sen Debasish , Oniskey Taylor , Kim Charlie , Krummel Matthew
Published: January 2023

Mechanisms responsible for pulmonary tissue homeostasis and regeneration are incompletely understood. This study investigated whether Trefoil factor family (TFF) proteins, regulators of gastrointestinal restitution, served any role in the pulmonary tract under steady state conditions or pathogen infection. At baseline, TFF2 lung mRNA transcript levels were highest compared to TFF1 and TFF3. Strikingly, TFF2 deficient mice (TFF2-/-) were hypoxic by 6-8 weeks of age, with abnormal alveoli, reduced blood oxygen levels, and defective integrin expression on alveolar type 1 cells. TFF2 was essential for mucosal barrier function in tracheal epithelia and TFF2 deficiency caused multiple transcriptional defects in adhesion, motility, and wound repair. Unexpectedly, TFF2 expression in hematopoietic cells was necessary for Type 2 immunity and recovery of pulmonary function after inoculation with the hookworm Nippostrongylus brasiliensis. TFF2 served a cell-intrinsic role in promoting macrophage polarization towards the M2 subset. Thus, in addition to epithelial restitution, TFF2 derived from hematopoietic cells instructs macrophage subset differentiation, promotes host immunity, and stimulates tissue repair within the pulmonary tract.
Superior protection and distinct phenotype of pulmonary CD8+ T cells elicited by mucosal vaccination (MUC5P.863)
Authors: Gilchuk Pavlo , Hill Timothy , Guo Yan , Clark Travis , Erickson John , Lu Pengcheng , Boyd Kelli , Williams John , Shyr Yu , Joyce Sebastian
Published: January 2023

CD8-positive T cells (CTLs) confer protective immunity against many infectious diseases, and thus the development of safe and efficient CTL-targeted vaccination is of high clinical significance. Although many respiratory pathogens invade the host through the lung mucosa, most licensed human vaccines elicit limited and systemic T cell responses, rather than strong local pulmonary responses. We therefore wondered whether elicitation of pulmonary CTL response with microbe-free CTL-targeted subunit vaccine might benefit the host and protect against respiratory infections. We discovered superior protection of humanized mice from lethal respiratory challenge with vaccinia virus with intranasal, but not intra-peritoneal, vaccination. Critically, this superior protection did not correlate with the magnitude of the systemic CTL response but was dependent on in situ elicitation of robust pulmonary epitope-specific CTLs, which were able to limit viral replication and prevent disease. Global transcriptome analysis revealed that pulmonary CTLs elicited by vaccination possessed distinct phenotypic and functional properties when compared to CTLs that homed to the spleen. To extend this finding, we developed an approach for eliciting protective CTL responses that utilizes nanoparticle-based mucosal delivery of epitope-targeted vaccine formulated with an optimal adjuvant.
A novel p65 inhibitor suppresses glutamate release and proliferation of macrophages: therapeutic implications for chronic neurological diseases. (THER3P.887)
Authors: Srinivasan Mythily , Blackburn Corinne , Lahiri Debomoy
Published: January 2023

Activated nuclear factor-κB (NFκB) plays critical role in the pathogenesis of chronic neurological diseases constituting an attractive therapeutic target. Based on the cross talk between NFκB and glucocorticoid signaling pathways, we developed a novel strategy to selectively target p65, the functionally critical subunit of NFκB. Glucocorticoid induced leucine zipper (GILZ) is a “divergence” hub linked to multiple proteins in the NFκB signaling pathway. Structurally, proline rich carboxy terminus of GILZ binds the transactivation domain of p65. An immunomodulatory peptide derived from the p65 binding region of GILZ, the GILZ-P ameliorated autoimmune disease in mice. Here we determine the secondary structural characteristics and investigate the efficacy of GILZ-P in lipopolysaccharide (LPS) activated ThP-1 macrophages used as model systems for human microglial cells. GILZ-P treatment did not adversely affect cell viability as determined by vital staining and flow cytometry. LPS stimulated mature macrophages treated with GILZ-P exhibited reduced proliferation and inflammatory cytokine secretion as assessed by the MTT assay and ELISA respectively. Mechanistically the inhibitory potential of GILZ-P was substantiated by the reduced nuclear translocation of p65 and decreased glutamate release. Collectively, our data support anti-inflammatory and neuroprotective effects for the PPII helical GILZ-P suggesting therapeutic applications in neuroinflammatory-neurodegenerative conditions.
Cbl-b deficiency results in abnormalities in both T cell sphingosine-1-phosphate receptor 1 function and response to the multiple sclerosis therapeutic agent, FTY720 (THER3P.881)
Authors: Fujiwara Mai , Anstadt Emily , Khanna Kamal , Clark Robert
Published: January 2023

FTY720 is a sphingosine-1-phosphate receptor 1 (S1P1) modulator used to treat Multiple Sclerosis (MS). While FTY720 is postulated to mediate its effect by enhancing T cell S1P1 internalization and degradation, the mechanisms underlying the therapeutic effect of FTY720 in MS are not fully understood. To further clarify the functional effects of FTY720 we utilized Cbl-b-deficient (Cbl-b-/-) mice. Cbl-b is an E3 ubiquitin ligase that regulates the PI3K-Akt pathway in T cells and its absence leads to resistance to regulatory T cells (Tregs) and multi-organ autoimmunity in mice. Given that MS has been associated with both SNPs in the CBLB gene and resistance to Tregs, Cbl-b-/- mice represent an important model for studying MS. We now report that Cbl-b-/- T cells demonstrate multiple functional abnormalities in S1P1. These include significantly less lymphopenia induced by the S1P lyase inhibitor, THI, (Cbl-b-/- blood CD4+ T cells decreased 19% vs 59% in wild type (WT) at 48 hrs), suggesting abnormal regulation of Cbl-b-/- T cell S1P1 in response to the endogenous ligand S1P. Paradoxically, Cbl-b-/- T cells demonstrate enhanced lymphopenia induced by the therapeutic agent FTY720 (Cbl-b-/- blood CD4+ T cells decreased 51% vs 26% in WT at 120 hrs). These results for the first time identify a complex role for Cbl-b in regulating expression and turnover of T cell S1P1 and suggest that SNPs in the CBLB gene may associate with a differential response to FTY720 in MS patients.
Senescence associated inflammasome (SAI) activation promotes cancer progression (TUM4P.909)
Authors: Guo Beichu , Zhang Jinyu
Published: January 2023

Oncogene-induced senescence functions as a barrier against cell transformation and tumorigenesis. Paradoxically, recent studies indicate that senescence also promotes tumor progression through secretion of inflammatory molecules or senescence-associated secretory phenotype (SASP). However, the signaling pathways leading to SASP and the interaction between senescent cells and innate immune cells are poorly understood. Utilizing human and murine breast cancer cells, as well as breast cancer mouse models, this study demonstrates that senescence-associated inflammasome (SAI) generate a positive feedback loop to propagate inflammation through interaction with innate immune cells, contributing to the tumor development. Our data show that cellular senescence induced by oncogenes, such as K-Ras12V and Her2/ERBB2, led to inflammasome activation and IL-1beta production in both murine and human breast cancer cells. Notably, we found that blocking inflammasome activity could suppress both IL-1beta production and oncogene-induced senescence in breast cancer cells. Our results further show that the crosstalk between senescent cells and innate immune cells promoted inflammation and breast cancer progression. Together, our findings demonstrate a critical role for the senescence-associated inflammasome in oncogenic inflammation and tumor development.
The myeloid receptor TREM2 exacerbates DSS-induced colitis and promotes colitis-associated cancer (TUM4P.916)
Authors: McVicar Daniel , Howard O.M.Zack , Sublesk Jeff , Quigley Laura , Yang Youfeng , Rayman Patricia , Rini Brian , Linehan W. Marston , Sayers Thomas , Finke James , Stappenbeck Thaddeus , Ford Jill
Published: January 2023

The Triggering Receptors Expressed on Myeloid cells (TREM) gene cluster contains receptors that regulate a variety of leukocytes. TREM1 synergizes with Toll Like Receptors to promote cytokine production in neutrophils, macrophages and dendritic cells (DC). Paradoxically, although TREM1 and TREM2 both signal via DAP12, TREM2 is a negative regulator of inflammation in macrophages and DC. TREM2 is expressed in the colon but its role in colitis and colitis-associated cancer (CAC) is poorly understood. We found TREM2 expressed in monocytic myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM). Moreover, TAM expressed a apparent TREM2 ligand. To test the role of TREM2 in inflammation-induced cancer we studied the azoxymethane (AOM)-dextran sulfate sodium (DSS)-induced model of CAC. DSS administration led a pronounced influx of myeloid cells and upregulation of both TREM1 and TREM2. Surprisingly, we found that Trem2-/- mice had less severe colitis as indicated by reduced histological scores, colon shortening and inflammatory cytokine production in the colon. AOM-DSS carcinogenesis in Trem2-/- mice resulted in slightly reduced total polyp counts and mitotic epithelial cells but a substantial reduction in the numbers of advanced carcinomas compared to wild type mice. Taken together, our data suggest an important role for TREM2 in the regulation of colitis and CAC likely via control of epithelial proliferation during colonic injury and inflammation.
Activation of IL-6/STAT3-signaling cascade induces suppression of antigen presentation by human dendritic cells (TUM4P.917)
Authors: Kitamura Hidemitsu , Ohno Yousuke , Ohtake Junya , Kaneumi Shun , Kishikawa Takuto , Terada Satoshi , Sumida Kentaro , Masuko Kazutaka , Kita Toshiyuki , Takahashi Norihiko , Taketomi Akinobu
Published: January 2023

Immunosuppression in tumor microenvironments is one of the critical issues for cancer immunotherapy. We have been demonstrated that IL-6, a pleiotropic cytokine that regulate the growth, differentiation and survival of a variety of cells, significantly inhibited maturation of murine dendritic cells (DCs) via STAT3 activation both in vitro and in vivo. In this study, we focused on IL-6/STAT3-signaling cascade in human dendritic cells (DCs), and investigated the effects of IL-6 on antigen-presenting ability of DCs. Surface HLA class I, HLA class II, and costimulatory molecules of human DCs were evaluated after the stimulation with IL-6 in vitro. As a result, HLA-DR and CD86 expressions were significantly reduced by IL-6 treatment of human DCs. The reduction was remarkably blocked in the presence of STAT3 inhibitor. In addition, IFN-γ production by T cells after TCR-stimulation was significantly suppressed in the presence of IL-6-treated DCs compared with control DCs. Moreover, we found that activation of antigen-specific CD4+ T cells by DCs was remarkably reduced by the IL-6-treatment. In this study, we demonstrated that IL-6/STAT3-signaling cascade was one of the regulating factors for antigen presentation of DCs. From these findings suggest that IL-6/STAT3 signaling cascade regulates immunosuppressive function of human DCs, which would be a promising target for improving the effects of cancer immunotherapy.