bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytoplasmic labile zinc regulates IL-1β production in human monocytes/macrophages via mTORC1-induced glycolysis
Authors: Kim Bonah , Lee Su Jeong , Kang Yeon Jun , Lee Ga Hye , Kim Hee Young , Lee Won-Woo
Published: January 2023

Zinc is an essential micronutrient that plays critical roles in multiple facets of biological processes, including growth, development, proliferation, and differentiation. Since dysregulated zinc homeostasis impairs overall immune function and resultantly, increases susceptibility to infection, much attention has been recently paid to immunomodulatory roles of zinc in immune cells. Rapid and efficient production of proinflammatory cytokines, such as IL-1β and TNF-α, is a primary effect function of monocytes/macrophages for host defense. However, the molecular mechanism involved with zinc-mediated modulation of cytokine production in monocytes/macrophages needs to be better understood. In present study, we provide evidence that cytoplasmic labile zinc is essential for modulation of IL-1β production in human monocytes/macrophages via mTORC1-induced glycolysis. We found that cytoplasmic level of labile zinc in human monocytes/macrophages was largely influenced by extracellular zinc concentrations, possibly via zinc importers such as ZIP1- and ZIP8-mediated influx. The phosphorylation of S6 kinase, a substrate of mTORC1, was significantly enhanced by zinc-mediated inhibition of PP2A, a phosphatase contracting S6K phosphorylation and consequently, IL-1β production was increased by activated mTORC1-induced glycolysis. These results provide new insight how labile zinc modulate a production of cytokines by metabolic reprograming in human monocytes/macrophages.
Bacterial Polyphosphates Modulate Macrophage Responses to Infection
Authors: Bosmann Markus , Roewe Julian , Stavrides Georgios , Marini Federico , Smith Stephanie A. , Reinhardt Christoph , Morrissey James H.
Published: January 2023

Polyphosphates are linear polymers of inorganic monophosphate residues (Pi) and these ancient metabolites exist in all living cells. In bacteria, polyphosphate kinases (PPK) and polyphosphatases (PPX) control the accumulation of polyphosphates from ATP as long chains (>300–1000 Pi) for energy storage and prokaryotic homeostasis.

Here, we demonstrate that bacterial (long-chain) polyphosphates bound and were internalized by mouse macrophages. Long-chain polyphosphates modulated the expression of more than 1,800 LPS/TLR4-regulated genes in macrophages. This interference by polyphosphates included antagonism of hundreds of interferon-regulated genes as explained by reduced interferon-β release and responsiveness, reduced phosphorylation of STAT1 and diminished major histocompatibility complex II. Polyphosphates also prevented the LPS/TLR4-induced induction of NOS2 and M1 polarization. In C57BL/6J mice infected with live E. coli, PPK deficiency or polyphosphate neutralization using recombinant PPX resulted in better survival rates, amplified macrophage recruitment and improved pathogen clearance.

In conclusion, bacterial polyphosphate metabolites interfere with macrophage responses and the manipulation of such interactions could help to restore immunity to infection.
Myeloid cells expressing CK2α modulates resistance to systemic Listeria monocytogenes infection
Authors: Larson Sandy R , Crisler Cris , Lenz Laurel L
Published: January 2023

Reversable phosphorylation is a key regulatory mechanism of nearly all cellular processes. Kinase activity driving phosphorylation has implications in cell growth, survival, development, differentiation, and importantly, immune responses to pathogens. The protein kinase CK2 (casein kinase II) is a ubiquitously expressed and highly conserved haloenzyme with over 500 direct target substrates identified. Because of this promiscuous activity, CK2 is extensively involved in immune cell function, described in both innate and adaptive immune cells. However, functionality of CK2 is not clearly delineated, largely due to the lack of murine models available for study. Engineered mice deficient in either the catalytic α or α′, or the regulatory β subunits are either non-viable or non-fertile. Therefore, we have generated a conditional murine knockout model of CK2α by cre-mediated excision in lysozyme M (LysM) expressing cells. Progeny are viable and fertile, with no deficiencies of immune cell populations. Using this model system, we have identified a role of CK2α in negatively regulating monocyte and neutrophil infiltration and endosome acidification during the bacterial Listeria monocytogenes (Lm) infection, correlating with uncontrolled bacterial replication. Further, decreased inflammatory myeloid cells in infected tissues is a cell-intrinsic effect of CK2α. Future work is aimed to understand the mechanisms by which CK2α negatively regulates the accumulation of immune cells in infected tissues. Additionally, we now have a tool for investigating in vivo effects of CK2α that we can use to extend beyond these initial studies to evaluate its role in other bacterial/viral infections, autoimmunity and even cancer.
Mycobacterium tuberculosis ESX-1 induces eicosanoid mediated neutrophil influx to the lung during Tuberculosis
Authors: SAQIB MOHD , Mishra Bibhuti B
Published: January 2023

Tuberculosis disease (TB) is characterized by excessive inflammation and irreversible tissue damage. To determine the bacterial factors responsible for regulating inflammation, different TB susceptible mouse strains were infected with a pool of transposon mutants of M. tuberculosis (Mtb) that revealed transposon insertions into ESX1 locus impaired the ability to cause disease in susceptible mice and elicit inflammatory response comparable to wild type Mtb. However, the underlying mechanisms of ESX1 mediated tissue damage in vivo is obscure. This study aims at elucidating the mechanism by which Mtb ESX1 regulate tissue damaging inflammation in vivo. Mtb ESX-1 secretion function was required for inducing neutrophil infiltration to the lungs, of TB susceptible mice (iNOS−/−, IL1R1−/−, C3HeB/FeJ). Deletion of ESX-1 from virulent Mtb (ΔESX-1) had little impact on T cells, but significantly reduced the neutrophil/T lymphocyte ratio that favored efficient bacterial control. Co-infection of these mice with wild-type and ΔESX-1, rescued the defective neutrophil influx seen in mice infected with the ΔESX-1 alone. Further, the neutrophil influx elicited by wild type Mtb follows the loss of pro-surfactant C expressing type II alveolar epithelial cells that coincided with the generation of lipoxygenase derived eicosanoids in the lungs; indicating involvement of these chemotactic lipid mediators in initiating neutrophil recruitment in an ESX1 dependent fashion. Taken together, this study provides evidence for the involvement of ESX1 in regulating tissue damaging neutrophilic inflammation during TB, expanding the current understanding of the pleiotropic functions of type VII secretion apparatus.
Vitamin D modulates innate immunity in pulmonary tuberculosis
Authors: Singh Archana , Panda Sudhasini , Tiwari Ambrish , Luthra Kalpana , Sharma Surinder Kumar
Published: January 2023

Vitamin D and vitamin D receptor (VDR) may play role in altering host defence against Mtb via cathelicidin production and regulating nitric oxide (NO) production through inducible nitric oxide synthase (iNOS). 100 active pulmonary tuberculosis (PTB) patients, 75 household contacts and 70 healthy controls were recruited. Serum Vitamin D and NO levels were measured using CLIA and colorimetry based kit respectively. VDR and iNOS mRNA was studied in monocytes using qPCR. Serum VDR, cathelicidin and iNOS levels were measured using ELISA. Lower serum levels of vitamin D was found in PTB group compared to household contacts and healthy controls (P<0.001). VDR (both mRNA and protein) and iNOS mRNA levels were also lower in PTB group compared to household contacts and healthy controls (P<0.005)probably due to decreased levels of vitamin D. However, despite low serum vitamin D, serum cathelicidin levels were higher in PTB group compared to household contacts and healthy controls (p<0.01) which could be attributed to direct trigger via toll like receptors where Mtb is multiplying actively. Serum iNOS and NO levels were higher in PTB group compared to household contacts and healthy controls (p< 0.05), despite decreased iNOS expression which needs further evaluation. Increased VDR and iNOS expression in household contacts compared to both PTB and healthy controls could be due to optimum levels of vitamin D along with some exposure to Mtb as evident by increased levels of cathelicidin and NO compared to healthy controls. Adequate levels of vitamin D, VDR and iNOS that in turn promote production of antimicrobial cathelicidin and NO might have protective role against TB among household contacts and needs further investigation for its therapeutic potential.
Genetic mechanisms contributing to IFNβ regulation of Lyme Arthritis: involvement of the Borrelia burgdorferi arthritis associated locus 1 (Bbaa1)
Authors: Li Jinze , Ma Ying , Paquette Jackie , Teuscher Cory , Weis Janis J
Published: January 2023

Lyme disease shows a spectrum of clinical disorders in patients that are infected with the tick borne spirochete Borrelia burgdorferi. Studies have shown that C3H mice develop severe arthritis while C57BL/6 (B6) mice display mild arthritis when infected with the same isolate of B. burgdorferi. In order to study the role of the host genetics in regulating the severity of Lyme arthritis development, we used forward genetics and identified a genetic region, Bbaa1, that includes the Type I IFN cluster on Chr4. We further developed congenic mice isolating the C3H allele of Bbaa1 onto the background of B6 mice and determined that Bbaa1 regulates arthritis severity by upregulating Type I IFN. Antibody blocking revealed IFNβ, but not IFNα, as the key effector that drives severe Lyme arthritis. Because there are no SNPs between the C3H and B6 IFNβ genes, we hypothesized that other genetic factors in the Bbaa1 locus regulate the differential expression of IFNβ and Lyme arthritis severity. RNA silencing was used to identify candidate genes within Bbaa1 that regulate IFNβ expression in myeloid cells. Cdkn2a was identified as a strong regulator of IFNβ expression. Although Cdkn2a encodes two proteins, p16 and ARF, only the silencing of Arf resulted in the suppression of IFNβ expression. Additionally, the overexpression of ARF led to enhancement of IFNβ. We propose that ARF regulates IFNβ by two pathways involving the proteins p53 and BCL6. Indeed, blocking of BCL6 by a small molecule inhibitor induced IFNβ activation in the joint tissue of B6 mice and enhanced the severity of Lyme arthritis. Future work will focus on the impact of the two pathways on IFNβ expression and arthritis severity in mice.
Pathogenic T cell subset Th17 is regulated by chromatin remodeling dependent manner to switch Th1 phenotype
Authors: Hashimoto Kahoko , Kobayashi Daichi , Oda Toshiki , Hasegawa Akihiro , Nakayama Toshinori
Published: January 2023

In many autoimmune disease such as type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease (IBD), it has been reported that pathogenic Th17 cells exhibit remarkable plasticity to classical Th1 phenotype at the site of autoimmune inflammation. This effecter Th17 cells is also called non-classical Th1 cells because they show the ability to produce IFNγ, similar to conventional Th1 cells, and also continue to produce IL-17 cytokine. The precise mechanism of transcriptional regulation in Th17/IFNγ cells is still not clear yet. By using the splenic naive CD4 T cells, cells were cultured under the Th17 condition including IL-6 and TGFβ for 3 days to 7 days and then cells were added IL-12 to provide Th1-like differentiation condition. In vitro induced Th17 cells were stimulated with anti TCR antibody and assessed the ratio of IL-17 protein production, RORγt and T-bet expression as protein level by flow cytometer. Then, induced Th17 cells were performed to the secondary culture under the several concentration of IL-12 and TGFβ to be able to detect the production of IFNγ, and T-bet. Also we analyzed the specific gene expressions of transcription factor by RT-PCR or quantitative PCR on each day. In this study, we focused on the plasticity of Th17 to Th1 and also compared to the effecter T cell function. We focused on the function of chromatin remodeling in Th17 cells producing both Il-17 and IFNγ by performing ChIP analysis of double producing Th subset and detected that this type plasticity is unique in pathogenic Th17 cell.
CTLA-4-dependent trogocytosis promotes the interactions between Tregs and antigen-presenting cells
Authors: TEKGUC MURAT , WING JAMES BADGER , OSAKI MOTONAO , LONG JIA , SAKAGUCHI SHIMON
Published: January 2023

Regulatory T cells (Tregs) are responsible for the maintenance of immune homeostasis. They express the coinhibitory receptor CTLA-4 in high quantities, whose loss causes severe systemic autoinflammation in both mice and humans. CTLA-4 limits the expression of costimulatory CD80/CD86 proteins by antigen-presenting cells (APCs). However, the details of this mechanism are not fully understood. When we analyzed in vitro cocultures of CD80- or CD86-GFP expressing murine dendritic cells (DCs) with ex vivo sorted Tregs, we found that mutant Tregs, whose CTLA-4 lacked the cytoplasmic tail necessary for its endocytosis function, could still uptake CD80- or CD86-GFP from DCs via CTLA-4-dependent trogocytosis. Moreover, the specific CD80/86 uptake by CTLA-4, also facilitated the indirect transfer of membrane lipid particles and other surface proteins. Both flow cytometry and confocal microscopy revealed that immune synapse formation between Tregs and APCs was enhanced depending on CTLA-4 expression by Tregs. Confocal microscopy also demonstrated that uptaken CD80/CD86-GFP proteins colocalized with LFA-1 capping, an indicator of stable immune synapses. Furthermore, in vivo delivery of CTLA-4 WT and knockout (KO) Tregs into CD45.1+RAG2 KO mice showed that transfer of congenic CD45.1 protein from host cells to donor Tregs was significantly diminished in CTLA-4 KO Tregs compared to WT Tregs. The tail portion of CTLA-4 was also found to be dispensable for CD80/86 downregulation and suppressive functions of Tregs. In conclusion, CTLA-4-promoted immune synapse formation provides a stable platform between Tregs and APCs, which results in both depletion of CD80/CD86 via trogocytosis and indirect uptake of other surface proteins from APCs.
Differential transcriptional response of plasmacytoid dendritic cells in mice with lupus
Authors: Baeza Carlos Wong , Mora Albany Reséndiz , Pensado Monserrat Figueroa , Aveleida Giovanna Barrera , Ángeles Karen Sánchez , Cortés Cinthya Portas , Gómez Eréndira Molina , Saldívar Carla Landa , Baeza Isabel Wong , Baeza Isabel
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune, chronic and multi-systemic disease. SLE patients present high levels of type I IFN, which is mainly produced by plasmacytoid dendritic cells (pDCs). In this work, we analyzed the gene expression of pDCs from mice with lupus induced by non-bilayer phospholipid arrangements (NPA). An increase in type I (alpha and beta) IFN was detected in the serum of mice immunized with NPA-bearing liposomes, compared with mice that received smooth liposomes or saline solution. Furthermore, 20 days after the beginning of the administration of the NPA-bearing liposomes, a statistically significant increase in the absolute number of pDCs was found in the spleen and in the mesenteric lymph nodes of mice administered with these liposomes, compared with mice administered with saline solution or with smooth liposomes. When we compared the gene expression of pDCs from mice with lupus with pDCs from mice that received smooth liposomes, 9 of the 22 over-expressed genes corresponded to the innate response and 13 to the adaptive response, while 35 of the 47 under-expressed genes corresponded to the innate response and 12 to the adaptive response. After analyzing the gene expression patterns, we propose that there may be three subsets of pDCs in the spleens of mice with lupus: one that produces high levels of type I IFN; a second one that produces pro-inflammatory and immuno-regulatory cytokines and thus participates in both innate and adaptive responses; and a third one that recognizes NPA-bearing liposomes and processes and presents them through the CD1 molecule.
The secretory pathway-resident SNARE Sec22b regulates nitric oxide and cytokine production in dendritic cells
Authors: Descoteaux Albert , Dion Renaud , Descoteaux Julien , Fabié Aymeric , Stäger Simona , Duque Guillermo Arango
Published: January 2023

Soluble NSF attachment receptor (SNARE) proteins regulate the vesicle transport machinery in phagocytic cells. Within the secretory pathway, Sec22b is an ER-Golgi intermediate compartment (ERGIC)-resident SNARE that controls phagosome maturation and function in macrophages and dendritic cells. The ERGIC controls the release of cytokines and may impact that of nitric oxide (NO), the latter of which is synthesized by the Golgi-active inducible nitric oxide synthase (iNOS). Whether the ERGIC SNARE Sec22b controls NO and cytokine secretion, is unknown. Using bone marrow-derived dendritic cells (BMDC), we demonstrated that iNOS colocalizes with ERGIC/Golgi markers, notably Sec22b and its partner syntaxin-5 (Stx5), in the cytoplasm and at the phagosome. Pharmacological blockade of the secretory pathway hindered NO and cytokine release, and inhibited NFκB translocation to the nucleus. Importantly, RNAi-mediated silencing of Sec22b revealed that NO and cytokine production were abrogated at the protein and mRNA levels. This correlated with deregulated mitogen-activated protein kinase (MAPK) signalling and reduced nuclear translocation of NFκB. Collectively, our data unveiled a novel function for the ERGIC, and its resident SNARE Sec22b, in the release of inflammatory mediators.
PKC signaling plays an essential role in dendritic cell differentiation
Authors: Chavel Colin A , Lee Kelvin P
Published: January 2023

Differentiation of progenitor cells into dendritic cells (DC) is a complicated process that requires multiple signaling cascades. Tumor-derived factors can inhibit this process in part through repression of protein kinase C isoform β (PKCβ) gene expression mediated by STAT3 activity. How PKC signaling exerts this differentiation response has not been fully characterized. We found PKC required both the NFκB and ERK pathways to induce DC differentiation, as examined through T-cell proliferation assays where either pathway was blocked by chemical inhibition. We uncovered novel interactions for PKC dependent activation of both the NFκB and ERK during DC differentiation. The canonical NFκB pathway was activated by PKCβ through the CARD11 complex via novel mechanisms, including bypassing the established role for both TAK1 and MALT1 in downstream IκB phosphorylation and a newly characterized mechanism for IKK activation. The non-canonical NFκB pathway was also activated in a PKCβ dependent manner outside of the typical ubiquitin dependent degradation cascades activated by TRAF complexes. The ERK pathway was activated through cRaf by multiple PKCs in part through inhibition of RKIP activity. Multiple ERK targets were activated, including RSK and Jun-Fos complexes, as well as an ERK dependent Foxo3a depletion not yet characterized in DC differentiation. Furthermore, we found crosstalk mechanisms between the three pathways that resulted in early or late activation of the pathways and was seen at both the gene and protein levels. Taken together, these results indicate PKC as an important signaling node for DC differentiation.
miR-146a −/− deficiency in the DC compartment results in increased effector phenotype CD4+ and CD8+ T cells in response to LCMV infection
Authors: Flynn Patrick A , Lind Evan
Published: January 2023

miR-146a is a micro-RNA that acts on transcripts upregulated in response to TLR induced signaling. Activation of TLRs results in maturation and survival of dendritic cells (DCs) to prime T cells. Interestingly, little is known how miR-146a regulates DC phenotype/function and whether it has direct effects on the regulation of T cell function. To probe this question, we utilized a miR-146a conditional knockout mouse model to study both DCs and lymphocytes. Our in vitro studies of DCs show that miR-146a KO BMDCs have increased uptake and processing of DQ-OVA, suggesting increased antigen uptake and processing. Additionally, miR-146a KO BMDCs had increased gMFIs of both CD80 and CD86, suggesting increased co-stimulatory potential. To test for differences in T cell activation and priming, we used the LCMV infection model to analyze T cell responses in the context of miR-146a KO DCs in vivo (miR-146a cKO). We found that miR-146a cKO mice showed increased cross-presenting XCR1+ cDC1s and XCR1+ CD103+ DCs compared to miR-146a competent mice. T cells from miR-146a cKO mice had increased CD44 and PD-1 double-positive effector phenotype. Naïve miR-146a cKO mice had increased Tcm-like CD4+ T cells. Naïve miR-146a cKO mice had increased Tcm-like and Tscm-like CD8+ T cells. Our data indicate that the lack of miR-146a results in a skewing of DC subsets to favor XCR1+ DC1 and XCR1+ CD103+ populations who are known to excel at cross-presentation and migrate through tissues. Our data also suggests that miR-146a cKO mice activate more effector phenotype T cells and increase the proportion of Tcm and Tscm-like T cell populations which result in more robust adaptive T cell responses.
Essential role of NPAT in regulating T cell development and function
Authors: Cui Zejin , Zhao Fan , Lu Linrong
Published: January 2023

The T-cell development process is precisely controlled at different stages. For example, cell proliferation during T cell development is limited to immature double negative (DN) thymocytes. Here we want to investigate the mechanism of stage-specific regulation of cell proliferation in this process. Nuclear protein ataxia-telangiectasia (NPAT) is an in vivo substrate of cyclin E-Cdk2 kinase and plays a critical role in coordinating histone gene expression during the G1/S-phase transition and to ensure S-phase entry, thus play a regulatory role in determining the proliferation level of different cells.

By the using of conditional gene deletion in thymocytes, we found that ablation of NPAT resulted in severe thymocyte development defect including reduced thymocyte number and developmental arrest from ISP to DP, indicating a critical role of NPAT in thymocyte development. We have also constructed a mouse strain that ablates NPAT expression in peripheral mature T cells. NPAT deficiency in peripheral T cells only resulted in a mild decrease in the number of CD4 and CD8 cells, while maintained normal T-cell homeostasis in peripheral lymphoid organs. However, the proliferative capacity of naïve CD4 or CD8 T cells in NPAT cKO mice were impaired upon activation, which correlated with less severe symptoms in experimental autoimmune encephalomyelitis (EAE) disease upon antigen-immunization. We are currently exploring the regulation mechanism of NPAT function in different cell stages and will test the possibility of targeting NPAT in treat autoimmune diseases.
Lactoferrin and TGF-β1 synergize to enhance differentiation of OVA-specific Foxp3+ iTregs
Authors: Jo Hyeon-Ju , Park Sun-Hee , Jang Young-Saeng , Park Hui-Won , Kim Tae-Gyu , Kim Pyeung-Hyeun
Published: January 2023

We recently found that lactoferrin (LF), like TGF-β1, enhanced Ag non-specific Foxp3+ iTreg differentiation and the two molecules synergized to express Foxp3. In the present study, we investigated effect of LF and TGF-β1 on Ag-specific Foxp3+ iTreg differentiation. Naïve CD4+ T cells isolated from OT-II mice were co-cultured with OVA323–339-loaded T cell-depleted splenocytes as APCs. OVA-specific Foxp3+ Treg population was increased by LF and TGF-β1 alone, and synergistically increased by combination of the two molecules. However, these increases were diminished by additional CD28 stimulation using anti-CD28 Ab. In parallel, blockade of B7 molecules using CTLA4-Ig markedly increased the Foxp3 expression. These results imply that lactoferrin alone or particularly along with TGF-β1 can cause naïve T cells to differentiate into Foxp3+ Tregs, greater under weak co-stimulatory signals.
Temporal, TCR signal strength, and Notch signaling requirements for γδ T-cell functional programming and execution in vivo
Authors: Chen Edward , Lee Christina , Wiest David , Zúñiga-Pflücker Juan Carlos
Published: January 2023

γδ T-cells are critical mediators of both protective and destructive immunity. In this study, we aimed to understand how ontogeny, T-cell receptor (TCR) signal strength, and Notch signaling dictate γδ T-cell functional programming and execution. We employed RBPJ-inducible (RBPJind) mice, which enabled control of the initiation and duration of Notch responsiveness in hematopoietic cells. Our results revealed that generation of γδ T-cell IL-17 producers occurred more readily during fetal life. However, while generation of lymph node IL-17 producers occurred exclusively during fetal life, generation of lung IL-17 producers still occurred postnatally. Using KN6-transgenic (KN6tg) and RBPJind mice, we observed that strong TCR signals programmed γδ T-cells towards the IL-4 fate. Conversely, weak TCR signals programmed γδ T-cells towards the IL-17 fate, and was required for the generation of innate-like IL-17 producers. Notch signaling played a role in promoting the IL-4 fate but, contrary to previous reports, did not play a role in dictating the IL-17 fate. To investigate the role of ontogeny and Notch signaling in the ability of lung γδ T-cells to execute their IL-17 function during infection, we employed an M. tuberculosis model using trehalose dimycolate (TDM). We show that fetal-derived γδ T-cells produced IL-17 in response to TDM while adult-derived γδ T-cells did not. Notch was dispensable for the fetal-derived lung γδ T-cells to produce IL-17, contrary to previous reports suggesting that Notch was required in the periphery for γδ T-cell IL-17 production. Taken together, this study revealed the precise temporal, TCR signal strength, and Notch signaling requirements for γδ T-cell functional programming and execution in vivo.
ACLY regulates CD4 T cell activation and differentiation
Authors: Dahunsi Debolanle , Wolf Melissa , Wellen Kathryn , Rathmell Jeffrey C
Published: January 2023

ATP citrate lyase (ACLY) is a transferase that catalyzes the cytosolic conversion of citrate and coenzyme A (CoA) to acetyl-CoA and oxaloacetate. ACLY has been shown to link glucose/mitochondrial (TCA) metabolism to cytosolic acetylation of proteins, nuclear acetylation of histones and fatty acid synthesis. T cells dramatically increase their metabolic activity upon activation and activated effector T cell subsets utilize distinct metabolic programs. It has been shown that small molecule inhibition of ACLY decreases histone acetylation and effector differentiation in T cells. Here, we examine the effects of T cell-specific genetic knockout of ACLY. ACLY deficient CD4 T cells were found to have lower proliferation and activation marker expression compared to WT. In vitro differentiation of CD4 T cells into Th1 and Th17 subsets was also reduced in the absence of ACLY. Future directions include comparing ACLY KO to small molecule inhibition and examining the metabolic and epigenetic consequences of ACLY deficiency in CD4 T cells in inflammatory disease models.
Apparent sterilizing immunity in BCG-immunized mice challenged with an ultra-low dose of Mycobacterium tuberculosis
Authors: Plumlee Courtney R , Duffy Fergal J , Gern Benjamin H , Delahaye Jared L , Cohen Sara B , Rustad Tige R , Aitchison John D , Sherman David R , Zak Daniel E , Gerner Michael Y , Urdahl Kevin B
Published: January 2023

Tuberculosis (TB) is a highly heterogeneous disease that develops in a subset of individuals infected with aerosolized Mycobacterium tuberculosis (Mtb). To improve upon the standard experimental mouse model, we infected mice with an ultra-low Mtb dose (ULD), consisting of 1-3 founding bacteria, which reflects the physiologic inoculum of humans. These mice exhibited a broad range of outcomes, with bacterial burdens ranging from <10 to ~106 CFUs in individual lungs. Furthermore, they exhibited well-circumscribed granulomas that share features with human granulomas. To monitor outcomes in live mice, we identified a blood RNA signature that correlated with lung bacterial burdens. Remarkably, this mouse-derived signature predicted Mtb infection outcomes across species, including risk of progression to active TB in humans. Given these improvements in the mouse model, we wondered whether ULD infection would provide a better platform for assessing vaccine-induced immunity. We now report that BCG-immunized mice, compared to unimmunized controls, exhibited a lower percentage of infected mice at late timepoints after ULD Mtb challenge. Interestingly, this difference was not observed at earlier timepoints. Taken together, these results suggest that some BCG-immunized mice, upon subsequent Mtb aerosol challenge, are capable of eradicating their initial infection, a finding that has not previously been observed in the TB mouse model. We are currently developing methods to verify initial infection prior to potential clearance and are also investigating immune correlates of protection.
VAMP3 as a negative regulator of Leishmania amazonensis infection and antigen cross-presentation
Authors: Seguin Olivier , Descoteaux Albert
Published: January 2023

To colonize host immune cells, the protozoan parasite Leishmania creates a parasitophorous vacuole (PV) that can be either individual and tight-fitting or communal, large and spacious depending on the parasite species. We aimed to investigate the role of SNAREs in both PV formation and parasite survival. Using bone marrow-derived macrophages (BMM) and confocal microscopy, we found that L. amazonensis communal PV recruits SNAREs and membrane from the plasma membrane (SNAP23), the recycling endosome (VAMP3), the endoplasmic reticulum (STX18) and the trans-golgi (Vti1a) with fusion with the lysosomes (LAMP1) while only the late endosomes (VAMP8) are accumulated to L. major individual PV without fusion with the lysosome. In the absence of VAMP3 we found that replication of L. amazonensis and PV volume roughly doubled. The V0 subunit of the V-ATPase has been proposed as a fusion promoter and as anti-inflammatory. More recently, the V0 subunit was shown to controls Leishmania PV biogenesis via cholesterol homeostasis. We then linked the PV volume augmentation to an augmented V-ATPase V0 subunit TCIRG1. We verified the inflammatory capacity of VAMP3 KO BMMs and found that NO production reduced. IFN-ɣ and LPS stimulated VAMP3 KO BMMs killing capacity was also halved during L. amazonensis infection. Furthermore, cholesterol homeostasis was also shown to play a role in antigen presentation and we found that VAMP3 KO BMDCs had augmented antigen cross-presentation capacities. In conclusion, we report that VAMP3 acts as a negative regulator of L. amazonensis replication by limiting cholesterol availability and through its role in limiting the availability of the V0 subunit of the V-ATPase.
CD4+ T cells modulates efficacy of microbiome transplantation in treatment of Clostridiodes difficile infection
Authors: Abt Michael C , Littmann Eric R , Denny Joshua E , Lee Jung-Jin , Bittenger Kyle
Published: January 2023

Clostridiodes difficile is an opportunistic pathogen that infects the large intestine following perturbation of the intestinal microbiome causing epithelial damage and debilitating, potentially fatal, colitis. Fecal microbiome transplantation (FMT) is a clinically proven therapy to treat recurrent C. difficile infection. Despite remarkable efficacy, implementation of FMT therapy is limited and the mechanism of action remains poorly understood. Here, we demonstrate a critical role for CD4+ T cells in supporting FMT success using a murine C. difficile infection system. Following FMT, chronically infected wild-type mice resolve C. difficile while cohoused, littermate Rag1−/− mice fail to clear the infection. FMT in C. difficile infected mice that lack B cells, CD8+ T cells or CD4+ T cells reveals a necessary role for CD4+ T cells, but not B cells or CD8+ T cells, in resolution of C. difficile following FMT. Analysis of intestinal bacterial communities demonstrate the microbiota of FMT-responsive mice assimilates toward the composition of the FMT donor, while the non-responsive Rag1−/− and CD4+ T cell deficient mice exhibit impaired FMT engraftment. Further, FMT does not restore secondary bile acid pools in the cecum of non-responsive mice, an important metabolite associated with FMT efficacy. Combined, these data support a role for host immunity in determining efficacy of microbiota-based therapeutics.
Vitamin C insufficiency aggravates dextran sulfate sodium (DSS)-induced colitis and colitis-associated colon cancer by regulating IL-6 and IL-22 production in Gulo(−/−) mice
Authors: Kim Yejin , Lee Dahae , Jo Hyejung , Kang Jae Seung
Published: January 2023

Mucosal damage in inflammatory bowel diseases (IBDs) involves the dysfunctional immunoregulation of the gut. Among immunoregulatory factors, oxidative stress is abnormally high level in IBDs, and their destructive effects may contribute to the initiation or propagation of the disease. Vitamin C has anti-oxidant, anti-inflammatory and immunomodulatory effects. So, we investigated the effect of vitamin C on dextran sulfate sodium (DSS)-induced colitis in Gulo(−/−) mice which cannot synthesize vitamin C. Vitamin C-insufficient Gulo(−/−) mice showed the increased oxidative stress and inflammation in the colon. The production of interleukin (IL)-22, the recruitment of NKp46(+) cells, and the activation of p38 MAPK were decreased in the vitamin C-insufficient Gulo(−/−) mice accompanied by decreased mucin-1 expression in compared with vitamin C-sufficient Gulo(−/−) mice and wild-type mice. Moreover, the production of IL-6 was increased, and STAT3 and Akt were activated, which involved the decreased expression of E-cadherin and granyhead like-2 (GRHL2). Consequently, vitamin C-insufficient Gulo(−/−) mice eventually go with severe colitis and cancer development. Therefore, our results suggest that vitamin C has a protective effect on DSS-induced colitis, and has a progressive role in the colon cancer by regulating the production of cytokines and the induction of inflammation.
Formation of mesenteric tertiary lymphoid organs shapes the immune cell and lymph trafficking from the intestine in a Crohn’s disease-like mouse model
Authors: Czepielewski Rafael Sanguinetti , Onufer Emily J , Erlich Emma , Kim Ki-Wook , Saunders Brian , Young Shannon , Kumar Shashi , Zinselmeyer Bernd , Hsieh Chyi-Song , Davis Michael J , Randolph Gwendalyn J
Published: January 2023

Current understanding of Crohn’s disease (CD) is that hereditary factors render the mucosal surface susceptible by either deregulated interactions between the bacterial flora and surface epithelial cells or an overwhelming host immune response. Given that lymph flow is essential for leukocyte trafficking and lipid absorption, understanding the effects of chronic inflammation in the intestinal-draining lymphatics is crucial for IBD treatment. Proper immune responses in the gut require immune cell migration towards secondary lymphoid organs via mesenteric collecting vessels. Our group described newly developed tertiary lymphoid organs (TLO) in mesenteric fat of CD biopsies, that are formed in association with lymphatic collecting vessels. However, mouse models that resemble lymphatic impairment and TLO formation in IBD are missing. Using a unique IBD model of Crohn’s disease-like ileitis (TNFΔARE mice) we demonstrate that these mice develop mesenteric TLO. Under TNFΔARE background, we generated a lymphatic reporter and a transgenic photoconvertible lines. Our results show that mesenteric TLO are integrated with collecting lymphatic vessels, obstructing leukocyte and lymph traffic from the intestine. Using adoptive transfer of microbiota-dependent TCR Tregs we found that TLO have the capacity to differentiate naïve T cells, yet they harbor tissue-resident T cells, demonstrating their duality. Strikingly, although anti-TNF therapy reduce ileum inflammation, TLO are resistant to the treatment, contributing to TNF therapy relapse or inefficacy. Our results demonstrate that chronic intestinal inflammation extend to the mesentery, impairing immune surveillance of the gut mucosal barrier, altering homeostasis.
Epithelial cell intrinsic IL-1R signaling regulates intestinal homeostasis and inflammation
Authors: Overcast Garrett , Eshleman Emily , Alenghat Theresa , Pasare Chandrashekhar
Published: January 2023

Intestinal epithelial cells (IECs) provide a critical first line of defense against microbes through barrier function, production of antimicrobial peptides (AMPs), secretion of cytokines, and recruitment of inflammatory cells. Whether single or multiple upstream cues regulate this diverse set of responses from IECs is not clear. Here we discover an unexpected role for Interleukin-1 Receptor (IL-1R) signaling in IECs in regulating intestinal homeostasis and inflammation. Specifically, absence of IL-1R in epithelial cells abrogates a homeostatic anti-microbial program including production of AMPs. Although IL-1R signaling alone is not sufficient to drive the AMP gene induction in IECs, the IL-22-driven anti-microbial program requires synergistic IL-1R signaling. Mechanistically, IL-1R signaling results in enhancement and temporal stabilization of STAT3 phosphorylation downstream of IL-22R. Thus, we reveal an unexpected cooperation between IL-1R and IL-22R in directly regulating an intestinal anti-microbial program. In contrast, absence of IL-1R in IECs protects mice from chemically-induced acute intestinal inflammation suggesting that IEC specific IL-1R signaling contributes to inflammation and pathology. We find that IL-1R signaling directly induces expression of chemokines as well as genes involved in the production of reactive oxygen species in IECs, a process that likely evolved to protect against enteric pathogens. Our findings establish a critical role for IEC specific IL-1R signaling in both steady state anti-microbial gene induction as well as intestinal inflammation. Further studies are likely to reveal potential targets for developing therapies against Crohn’s Disease and Ulcerative Colitis.
Human T cell immunity in barrier sites is phenotypically distinct but clonally connected to other sites
Authors: Poon Maya ML , Meng Wenzhao , Dogra Pranay , Lam Nora , Szabo Peter A , Kubota Masaru , Matsumoto Rei , Rahman Adeeb , Luning Prak Eline T , Farber Donna L
Published: January 2023

The gut, lung, and skin are physical and immunological barriers that serve as the first line of defense against pathogens and foreign substances. Due to sampling limitations, previous studies have been limited to studying human barrier sites in isolation and often in the context of specific diseases. Our donor tissue resource established in collaboration with LiveOnNY offers a unique opportunity to examine human barrier T cell immunity as a network and elucidate how barrier sites are connected to each other and to lymphoid sites (lymph nodes, bone marrow, spleen). Here, using cytometry by time-of-flight (CyTOF) and T cell receptor (TCR) CDR3b sequencing, we investigate the unique defining characteristics and connectivity of T cells in barrier tissue – gut, lung, and skin – to lymphoid sites and blood. Through high-dimensional marker analysis by CyTOF, our results show that T cell subset composition and phenotype across 8 different sites comprise 33 clusters that are highly conserved between donors of different ages (22–70 yrs). Skin and gut T cells are distinct from each other and from lung, lymphoid sites, and blood, while lung T cells share features with lymphoid sites and blood. TCR repertoire analysis shows that, though majority of gut and skin T cell clones are unique to the tissue, expanded clones within these barrier sites share clonal overlap with other barrier and lymphoid sites. Moreover, T cells in the lung exhibit increased sharing with lymphoid sites and blood. Together, these results reveal how human T cells are compartmentalized in the skin and gut through site-specific adaptations while demonstrating that gut, lung, and skin barrier T cells are connected through shared specificities in a network of immune protection across the body.
Lupus pathogenesis and autoimmunity are exacerbated by high fat diet-induced obesity in mice
Authors: Zhang Xin , Meng Juan , Shi Xuhua , Ali Hiba , Hellmers Linh , Mathew Neeha , Washington Talon , Quinet Robert , Davis William , Zakem Jerald , Keshavamurthy Chandana , You Zongbing
Published: January 2023

Objectives

System lupus erythematosus (SLE)patients have increased prevalence of metabolic syndrome. Our previous data showed that circulating Tfh (Tfh) cells were increased in SLE patients and correlated to their disease activities. Here, we investigated the pathophysiologic link between fat-diet induced obesity and SLE and underlying mechanism using MRL/lpr lupus prone mice.

Methods

Twenty MRL/lpr mice were fed with a regular diet (RD) or high fat diet (HFD, 60% calories comprised of fat). Body weight and skin lesion were recorded weekly. Urine protein using Bradford assay was assessed. Blood was collected for IgG, anti-dsDNA and anti-nuclear antibody (ANA) detection. At the end of experiment of week 14, kidney and skin biopsy were embedded in paraffin for H&E, PAS, and Masson’s staining to detect lupus histopathological lesions and quantified as kidney index based on fibrinoid necrosis, periglomerular infiltrate, and inflammatory dermatitis. Spleen paraffin specimens were stained for Tfh cells markers.

Results

Obesity was achieved with a significant difference of mouse body weight in HFD groups by week 3 (p<0.01). The aggravated SLE pathogenesis in HFD mice was characterized by skin rash, proteinuria, IgG glomerular deposition with increased histopathology index in kidney, and splenomegaly (p<0.05). Interestingly, increased of Tfh cells were found in the spleen of HFD group.

Conclusion

Our results demonstrated that HFD-induced obesity with increase Tfh cells contributes to the acceleration of SLE progression and metabolic abnormalities. Changing eating habit or interventions reducing obesity could improve both SLE symptoms and metabolic inflammation, especially in genetically predisposed individuals.
Therapeutic Benefit of Regulating Iron Metabolism in Spontaneous Lupus Nephritis
Authors: Scindia Yogesh , Wlazlo Ewa , Ghias Elizabeth , Voi Valentina , Cechova Sylvia , Leeds Joseph , Swaminathan Sundararaman
Published: January 2023

Background

Lupus nephritis (LN) is an end-organ complication of Systemic lupus erythematosus and is more common in premenopausal women. Hepcidin, the master regulator of iron homeostasis, modulates inflammation and is negatively regulated by estrogen. Therefore, we hypothesized that exogenous hepcidin may reduce the severity and delay the onset of LN.

Methods

Pre-nephritic 8-week-old or nephritic 16-week-old MRL/lpr female mice were injected Hepcidin (50ug, i.p) or vehicle twice a week and markers of renal injury and inflammation were examined at 18 and 20 weeks of age. The direct effect of hepcidin on macrophages was studied in-vitro.

Results

Hepcidin reduced intrarenal iron accumulation, and increased H-ferritin. This was associated with reduced renal inflammation and immune cell infiltration, which collectively mitigated microalbuminuria and tubular injury, independent of immune complex deposits and autoantibodies. The increase in H-ferritin, was associated with a reduced number of renal Ki-67+-F4/80+ macrophages. In-vitro, hepcidin induced H-ferritin in macrophages and reduced labile (Fe2+) iron. H-ferritinhi macrophages proliferated less upon Mcsf-1 stimulation and secreted less IL-1b, and IL-6 upon TLR-3 activation. Hepcidin reduced microalbuminuria when administered to nephritic, 16-week-old mice without worsening lupus-associated anemia.

Conclusions

We have identified that hepcidin targets iron homeostasis and reduces cardinal pathogenic features of LN. Importantly, Hepcidin treatment ameliorates kidney disease in mice with established proteinuria. Thus, our data highlight that targeting cellular iron metabolism with hepcidin represents a promising and a new therapeutic strategy in LN.
Endogenous IL-33 contributes to Treg expansion induced by IL-2/CD25, a novel IL-2 biologic developed for low-dose IL-2 therapy
Authors: Lui Jen Bon P , Malek Thomas R
Published: January 2023

Low-dose IL-2 therapy is being advanced in patients with autoimmunity by its ability to increase Tregs. A drawback of IL-2, however, is its short-half life and potential off-target immuno-stimulatory effects. To circumvent these limitations, we developed a longer-lasting mouse IL-2/CD25 fusion protein with greater selectivity toward Tregs. IL-2/CD25 is more effective than an equivalent amount of IL-2 in controlling diabetes of NOD mice. Furthermore, compared to other tissues, IL-2/CD25 more readily expanded pancreatic Tregs, suggesting a co-factor that acts with IL-2/CD25. We considered IL-33 as a possible candidate as it also promotes Treg expansion. In this regard, low-dose IL-2/CD25 selectively induced the IL-33R (ST2) on NOD pancreatic Tregs. Correspondingly, ST2 was not substantially induced in Tregs with impaired IL-2R signaling. More importantly, IL-33 supported the expansion of wild-type Tregs, but not Tregs with impaired IL-2R signaling. Thus, Treg expansion by IL-33 depends on IL-2. Reciprocally, Treg expansion in the pancreas by IL-2/CD25 required IL-33 because this expansion was suboptimal in IL-33-deficient B6 mice but was maximal when IL-33 was sequentially administered with IL-2/CD25. However, IL-2/CD25 and IL-33 did not act synergistically for Treg expansion in the pancreas of NOD mice, as IL-33 or IL-2/CD25 were similarly effective individually. This result may reflect that other inflammatory mediators or co-factors associated with the NOD pancreas also influence the Treg response to IL-2/CD25 and IL-33. Overall, these data indicate that tissue-derived IL-33 may contribute to the overall outcome of low-dose IL-2 therapy by supporting Treg expansion during therapy of autoimmune diseases.
Pharmacological activation of protein tyrosine phosphatase SHP-1 ameliorates autoimmune arthritis in mice
Authors: Markovics Adrienn , Toth Daniel M , Glant Tibor T , Mikecz Katalin
Published: January 2023

Background

The Src homology region 2 domain-containing phosphatase-1 (SHP-1) is known to exert negative regulatory effects on immune cell signaling. We observed previously that mice overexpressing an SHP-1 transgene were resistant to the development of autoimmune arthritis and showed decreased immune responses in the cartilage proteoglycan (PG)-induced (PGIA) model of rheumatoid arthritis (RA).

We sought to explore if pharmacological activation of SHP-1 in wildtype (WT) mice prevents or ameliorates arthritis.

Methods

To induce PGIA, mice were immunized with human (PG) in adjuvant. For in vivo SHP-1 activation, mice were treated with regorafenib (BAY 73-4506) per os either before arthritis symptoms developed (preventive treatment) or shortly after the initial signs of arthritis (therapeutic treatment). Regorafenib administration was continued for at least 21 days while arthritis incidence and severity were monitored.

Results

Preventive regorafenib treatment of WT mice significantly decreased arthritis severity (10 or 20 mg/kg) and incidence compared to vehicle treatment. Preventive treatment (10 mg/kg) increased tyrosine phosphatase activity of spleen cells and decreased IFNγ secretion by splenic T cells in vitro. Therapeutic treatment with 15 mg/kg decreased the severity of arthritis and lowered IFNγ production.

Conclusion

Our studies are the first to investigate the role of SHP-1 in autoimmune arthritis. We found that pharmacological activation of SHP-1 in vivo prevented the development of autoimmune arthritis or ameliorated arthritis severity when applied after the disease onset in the PGIA model of RA. Activation of SHP-1 might open a new therapeutic perspective in RA treatment.