bluuebunny/crossref_metadata_2025 huggingface dataset:
PGAM1 hypomethylation drives aerobic glycolysis in CD4 T cells to facilitate the host defense against tuberculosis
Authors: Chen Xinchun , Zhu JiaLou , Zhu Chuanzhi , Gao Fei , Cai Yi
Published: January 2023

Background

DNA methylation is important epigenetic mechanism that mediates cellular development and function, but whether DNA methylation is involved in regulating CD4 T cells in the context of tuberculosis (TB) is unclear.

Results

We used oxidative bisulfite sequencing to measure global DNA methylation levels in isolated CD4 T cells from patients with tuberculous pleuritis (TP), latent TB infection (LTBI) and healthy controls (HC). Compared to peripheral blood mononuclear cells (PBMCs), CD4 T cells from pleural fluid mononuclear cells (PFMCs) showed a significant different global DNA methylation profile, with much lower 5-methylcytosine levels in PFMC. A signature of 376 differentially methylated regions could discriminate between TP PBMC and PFMC T cells, including hypermethylated LAPTM5, FAM134B and FOXP1, and hypomethylated CCL5, TNF and AIM2 in PFMCs. KEGG pathway analysis showed that numerous metabolic pathways, especially ‘Carbon metabolism’, were highly enriched among the differentially methylated genes with lower methylation levels in TP PFMCs. Hypomethylation of the glycolytic enzyme PGAM1 promoted gene expression and increased aerobic glycolysis in CD4 T cells to permit CD4 T-cell proliferation and effector function.

Conclusions

Diminished DNA methylation levels —especially in genes involved in metabolism —are an important CD4 T-cell signature at the site of infection in TB. DNA hypomethylation of PGAM1is critical mechanism in regulating the CD4 T-cell immune response by enhancing aerobic glycolysis. These findings provide novel insights into the epigenetic regulation of CD4 T-cell responses and identify potential targets for immunotherapy in TB.
Invariant NKT cells functionally link microbiota-induced butyrate production and joint inflammation
Authors: Lee Sangsin , Kim Hye Young , Chung Doohyun
Published: January 2023

Emerging evidence indicates that the gut microbiota contributes to the regulation of joint inflammation by modulating the function of immune cells. However, the mechanism by which the microbiota regulates joint inflammation is unclear. To address this, we investigated the effect of the gut microbiota on antibody-induced arthritis (AIA). Feeding mice a high-fiber diet attenuated AIA in a microbiota-dependent manner. Among the short-chain fatty acids produced by the microbiota, butyrate suppressed cytokine production by invariant (i)NKT cells by inhibiting class I histone deacetylases. Furthermore, butyrate ameliorated AIA in wild-type, but not iNKT cell-deficient Jα18-knockout (KO), mice. Adoptive transfer of butyrate-pretreated iNKT cells had no effect on AIA in Jα18 KO mice, whereas transfer of untreated iNKT cells into Jα18 KO mice restored AIA. In conclusion, our data indicate that gut microbiota-induced butyrate production attenuates AIA by inhibiting cytokine production by iNKT cells. Thus, the microbiota–butyrate–iNKT cell axis may be a therapeutic target for joint inflammation.
Intrinsic function of STAT3 in B cells is to promote B cell proliferation and maturation
Authors: Oladipupo Favour , Egwuagu Charles E , Yu Cheng-Rong , Choi Jin Kyeong
Published: January 2023

We previously showed that STAT3 promotes T cell survival by restraining unbridled activation and proliferation of T cells (J Biol Chem. 286:30888–97). We also showed that STAT3 regulates Th17 differentiation and the loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune diseases (J Immunol. 180:6070–6). However, the intrinsic and extrinsic functions of STAT3 in B cells are not well understood. In this study, we have generated mice lacking STAT3 in CD19+ B cells and investigated the functions of STAT3 in B cells. Targeted deletion of stat3 in the conditional STAT3KO mouse strain (CD19-CRE-STAT3KO) was verified by Western blot analysis by showing loss of STAT3 expression in CD19+ B cells. We further show by lymphocyte proliferation assays and cell cycle analysis that the CD19-STAT3KO mice proliferated less compared to WT counterparts. Moreover, compared to the development of WT B cells, the STAT3KO mice contained a significantly higher percentage of immature B cells. These results suggest that STAT3 might exhibit diametrically opposite effects in B and T cells; Whereas STAT3 promotes B cell proliferation, STAT3 promotes T-cell survival and inhibits T cell activation/proliferation through up-regulation of Class O Forkhead Transcription Factors. In summary, our study reveals for the first time that STAT3 is required for B cell maturation.
Innate signaling pathways contribute towards formation of the extra-follicular B cell response
Authors: Lam Jonathan , Baumgarth Nicole
Published: January 2023

B cell responses are often assessed by the strength of the induced germinal center response. Yet proliferation and differentiation of antigen-specific B cells into antibody-secreting cells (ASCs) in extrafollicular (EF) responses, not germinal centers, are correlated in time with clearance from influenza virus infection. The signals responsible for initiation of the EF response, and subsequent differentiation into plasmablasts and plasma cells, have yet to be elucidated. Here we demonstrate that components of major innate signaling pathways are necessary for robust formation of ASCs during primary influenza infection. Of many different knockout mouse strains that lacked signaling in inflammatory pathways, only MyD88/TRIF double knockouts (DKOs) showed drastically reduced plasmablast/plasma cell formation as defined by expression of IRF4, a necessary transcription factor for ASC differentiation, and CD138, a marker of plasma cells. This was also demonstrated in DKO B cell chimeric mice, along with reduced influenza-specific antibody secretion. MyD88 and TRIF are downstream signaling components of Toll-like receptors (TLRs). However, mice lacking Unc93, TLR2 and TLR4 and thus all TLR signaling, but with MyD88 and TRIF intact, generated normal EF responses. We conclude that MyD88 and TRIF play a TLR-independent and B cell intrinsic role in early ASC generation. Current work is aimed at elucidating the role of MyD88/TRIF in the regulation of IRF4-induction.
Linking Endoplasmic Reticular Stress, ELL2, and Alternative Splicing
Authors: Milcarek Christine
Published: January 2023

RNA splicing patterns in antibody-secreting cells are shaped by endoplasmic reticulum stress, ELL2 transcription elongation factor induction, and changes in the levels of snRNAs. Endoplasmic reticulum stress induces the unfolded protein response; among these stress proteins is Ire1, whose auto-phosphorylation drives it to acquire a regulated mRNA decay activity. The mRNA-modifying function of phosphorylated Ire1 non-canonically splices Xbp1 and yet degrades other cellular mRNAs with related motifs, in a cell-specific manner. Naïve splenic B cells will activate Ire1 phosphorylation early on after lipopolysaccharide (LPS) stimulation, within 18 h; large-scale changes in mRNA content and splicing patterns result. Inhibition of the mRNA-degradation function of Ire1 is correlated with further differences in the splicing patterns and a reduction in the mRNA factors for snRNA transcription. Some of the >4000 splicing changes seen at 18 h after LPS stimulation persist into the late stages of antibody secretion, up to 72 h. Meanwhile some early splicing changes are supplanted by new splicing changes introduced by the up-regulation of ELL2 which is necessary for immunoglobulin secretion; it does this by changing mRNA processing patterns of immunoglobulin heavy chain and >5000 other genes. unexpectedly, ubiquitination of c-myc by the ELL family members also plays a role in plasma-blast survival.
Reciprocal regulation of Th2 and Th17 cells by PAD2-mediated citrullination
Authors: Sun Bo , Chang Hui-Hsin , Salinger Ari , Tomita Beverly , Bawadekar Mandar , Shelef Miriam A. , Weerapana Eranthie , Thompson Paul T. , Ho I-Cheng
Published: January 2023

Dysregulated citrullination, a unique form of post-translational modification catalyzed by the peptidylarginine deiminases (PADs), is associated with several major risk factors of rheumatoid arthritis, a disease characterized by the presence of anti-citrullinated protein antibodies. While PADs have been identified decades ago, their physiological roles in the immune system are still poorly understood. Hereby we report that PAD2 is the dominant PAD in Th cells. Its expression is induced by anti-CD3 stimulation. Pharmacological inhibition or genetic ablation of PAD2 augments Th2 differentiation but attenuates Th17 differentiation of human and mouse primary Th cells. This effect is due to alterations in the activity of GATA3 and RORgt. Mechanistically, both GATA3 and RORgt interact with and are citrullinated by PAD2 in primary Th cells. Citrullination of R330 of GATA3 weakens its DNA binding ability, whereas citrullination of four arginine residues of RORgt enhances its DNA binding. Furthermore, PAD2-deficient mice are more susceptible to allergic airway inflammation. Taken together, PAD2-mediated citrullination reciprocally regulates the differentiation of Th2 and Th17 cells.
VSTM4 is a novel negative regulator of T cell activation
Authors: Wang Jinghua , Manick Brian , Renelt Mark , Suin Jing , Hansen Louis , Person Anthony , Kalabokis Vassilios , Wu Guoping
Published: January 2023

The B7-like proteins play critical immunomodulatory roles and constitute attractive targets for the development of novel immunotherapies. In this study, we demonstrated that a fusion protein, containing the extracellular domain of V-set and transmembrane domain containing 4 fused to Fc (VSTM4-Fc), inhibited human T-cell activation. VSTM4 markedly suppressed human T cell proliferation in the presence of T-cell receptor signaling. Furthermore, VSTM4 significantly reduced IFN-γ, IL-2, and IL-17 cytokine production by human T cells. Moreover, VSTM4 profoundly decreased T cell activation marker CD69 expression on CD3+, CD4+, and CD8+ T cells. VSTM4 also inhibited naïve CD4+ T cell differentiation into Th1 cells. Our results showed strong binding of VSTM4-Fc to human activated T cells but weak binding to resting nonactivated T cells. Thus, we identify VSTM4 as a novel immunoglobulin superfamily (IgSF) protein with robust T cell inhibitory activity. VSTM4 represents a new negative regulator of T cells and may be a unique target for developing new immunotherapy strategies for the treatment of immune-related diseases, including autoimmunity and cancer.
CTCF is necessary for CD8+ effector T cell differentiation
Authors: Quon Sara J , Yu Bingfei , He Zhaoren , Russ Brendan , Turner Stephen J , Murre Cornelius , Goldrath Ananda W
Published: January 2023

Naive T cell differentiation into effector and memory subsets in response to infection requires the coordination of numerous factors that regulate changes in gene expression to support proliferation and cell function. Chromatin accessibility and DNA looping influence transcription factor binding, facilitate enhancer-promoter interactions, and insulate genes. CCCTC-Binding Factor (CTCF) is a highly conserved protein important for organizing the genome and partnering with transcription factors, but its role in CD8+ T cell differentiation has yet to be determined. We show that loss of CTCF in CD8+ T cells alters the expression of genes important for subset differentiation and these changes are reflected during Listeria infection where the formation of memory-precursor cells is favored while the formation of terminal effector cells is impaired. At a memory timepoint, CTCF knockdown enhances intestinal Trm accumulation despite impairing circulating memory maintenance. ChIP-Seq and HiC reveals that terminal effector cells gain CTCF binding within topologically associated domains, often overlapping with H3K27ac peaks which marks active enhancers and promoters. As genes near the overlap of CTCF and H3K27ac peaks have increased expression in terminal effector cells, we propose that one role of CTCF is to promote effector function by stabilizing promoter-enhancer interactions which influences the expression of fate-determining genes. Insights into the role of CTCF in CD8+ T cell differentiation during infection will yield valuable insight into dynamic chromatin changes regulating T cell differentiation and inform the development of therapies which better utilize the T cell response.
Epithelial versus myeloid-derived IL-33 controls different aspects of pathogen-specific immunity
Authors: Herbert DeBroski R , Herbine Karl , Hung LiYin , Pastore Christopher , Singh Brenal , Tanaka Yuki , Bryce Paul J. , Kambayashi Taku
Published: January 2023

Sensing of tissue injury through interleukin 33 (IL-33) release from damaged epithelial and endothelial cells directs effector function(s) of T helper and cytotoxic subsets, group 2 innate lymphocytes, and various myeloid cells. However, it is unclear whether noncanonical IL-33 production from conventional dendritic cells (cDC) shapes infection outcome. This study interrogated whether IL-33 from epithelial cells functioned differently from IL-33 derived from conventional dendritic cells (cDC) during helminth driven Type 2 inflammation. Data show mice temporally deleted of intestinal epithelial cell IL-33 (VillinCre-ERIL33flox/flox)failed to induce ILC2 expansion and host immunity against the hookworm parasite Nippostrongylus brasiliensis, whereas selective loss of cDC-derived IL-33 (CD11cCreIL33flox/flox) abrogated ST2+Foxp3+Treg responses, enhanced TH2 and ILC2 responses, and accelerated worm clearance. Lack of cDC intrinsic IL-33 impaired Foxp3+Treg expansion and anti-IL-2/IL-2 complex administration restored Treg numbers and suppressed worm clearance in CD11cCreIL-33flox/floxmice compared to CD11cCrecontrols. Collectively, this work emphasizes cellular source as a key determinant in the ability of IL-33 to direct pathogen-specific immunity.
The severities of Pseudomonas aeruginosa infection are enhanced by group 2 innate lymphoid cells in severely burned mice
Authors: Ito Ichiaki , Finnerty Celeste C , Herndon David N , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Pseudomonas aeruginosa burn wound infection and subsequent sepsis cause high mortality of severely burned patients. In our previous studies, group 2 innate lymphoid cells (ILC2) were increasingly detected in the lamina propria of severely burned mice. In this study, a role of ILC2 on the severity of pseudomonal systemic infections was investigated in severely burned mice. Severely burned mice (3rd degree, 25% TBSA burn) were infected s.c. with 103 CFU of P. aeruginosa. These mice were, then, treated i.p. with 100 μg/mouse of SR3335, an inhibitor of ILC2 proliferation, once daily for 2 days starting 1 day after burn injury. To test a role of circulating ILC2 on the pseudomonacidal activity of normal mouse PBMCs, these 2 cell preparations were transwell-cultured. In the results, the number of ILC2 increased in the circulation of mice 3–5 days after burn injury, but not in these burn mice treated with SR3335. All of the burn mice died within 6 days of P. aeruginosa infection, while 75% of the same mice treated with SR3335 survived. The pathogen was killed effectively by normal mouse PBMCs stimulated with heat-killed bacteria. The killing activity of these PBMCs was not shown in transwell-cultures with burn mouse PBMCs or circulating ILC2 isolated from severely burned mice. In contrast, normal mouse PBMCs transwell-cultured with burn mouse ILC2-depleted PBMCs killed the pathogen. These results indicate that the severities of P. aeruginosa infections are enhanced by ILC2 detected in circulation of burned mice.
Characterization of the function of murine guanylate-binding protein 9 in the context of bacterial and parasitic infections
Authors: Lichte Jens , Degrandi Daniel , Lindenberg Valesca , Mölleken Katja , Hegemann Johannes , Pfeffer Klaus
Published: January 2023

Interferon-γ (IFNγ) signaling induces high expression of immunomodulatory molecules in most cell types. Among these molecules, the members of the murine guanylate-binding protein (mGBP) family comprise a group of eleven proteins (mGBP1-mGBP11) shown to have important functions in cell-autonomous immunity. Upon IFNγ stimulation, mGBPs localize in vesicle-like structures distributed within the cytoplasm. Previous studies have shown that after infection with several intracellularly replicating pathogens, mGBPs rapidly relocate towards the pathogen-containing vacuoles (PCV), such as the parasitophorous vacuole (PV) of Toxoplasma gondii or the inclusions of Chlamydia trachomatis. Accumulation of mGBP2 at the PV membrane leads to loss of membrane integrity and subsequent restriction of parasite replication. Although mGBP proteins share a high sequence identity, differences in localization frequencies could be detected. For instance, mGBP1 and mGBP2 show the highest frequencies of recruitment to both Toxoplasma and Chlamydia PCVs. However, mGBP9 recruitment was more frequent to chlamydial inclusions than to T. gondii PVs. Thus, the importance of individual mGBPs might vary depending on the type of the pathogen. To analyze these findings in more detail, we started with the generation of mgbp9−/− cell- and mouse lines. Therefore, the CRISPR/Cas9 gene editing system with homologous directed integration of a single-stranded oligodesoxynucleotide is used to generate mGBP9 deficient ES cells for mouse and cell line development. In-depth analyzes of mgbp9−/− cells and mice in T. gondii and C. trachomatis infection will lead to a better understanding of immunity against intracellular pathogens.
Female mice express increased complement regulators and decreased intestinal ischemia-reperfusion injury
Authors: Fleming Sherry D , Ferm Jonathan , Amrein Jennifer
Published: January 2023

Intestinal ischemia reperfusion (IR) in male mice results in local and systemic tissue damage due to excessive complement activation and neutrophil infiltration. Complement components differ by sex in animal models of kidney and heart ischemia. In addition, estrogen alters blood vessel diameter during reperfusion and testosterone blockade attenuates nitric oxide production. Clinical studies associated oral contraceptive use with intestinal ischemia. Together, these data suggest that hormonal differences in females may change the mechanisms of intestinal IR injury compared to males. We hypothesized that increased intestinal complement regulator expression in females delays IR-induced C3 deposition compared to males. Male and female C57Bl/6 mice were subjected to Sham treatment or 30 mins intestinal ischemia followed by two hours reperfusion prior to collecting the mid-jejunum and serum. Intestinal sections were analyzed by RT-PCR for complement regulator expression. Additional analyses examined markers of cell death, complement deposition, and LTB4 production. Compared to male mice, female mice sustained delayed intestinal injury and decreased complement C3 deposition. In response to IR, female mice significantly increased autophagy markers and intestinal LTB4 secretion. Sham treatment increased mRNA of complement regulators, CRRY and CD55 in female mice while Factor H increased in response to IR in both sexes. Together these data suggest that local control of complement activation, neutrophil infiltration and autophagy in response to intestinal ischemia/reperfusion varies between sexes and may dictate distinct therapeutic intervention.
Dysregulation of G-CSF and neutrophil production is associated with chronic inflammation in elderly rhesus macaques
Authors: He Ziyuan , Takahashi Naofumi , Fahlberg Marissa , Slisarenko Nadia , Didier Elizabeth , Kuroda Marcelo
Published: January 2023

Aging is characterized with a loss of hematopoietic tissue in bone marrow, systemic chronic inflammation, and higher susceptibility to infectious diseases. We previously showed the tightly regulated kinetics and massive daily production of neutrophils during homeostasis in rhesus macaques aged 3 to 19 years old. Here we extended that study to further investigate the effect of aging on kinetics and production of neutrophils in more elderly rhesus macaques. We observed a neutrophil kinetics shift and higher in-group variability in rhesus macaques above 20 years old (equivalent to 70 years or more in humans) by in vivo BrdU pulse-chase labeling. In a cross-sectional study, we also measured complete blood cell counts and plasma cytokine levels from a group of 126 outdoor-housed captive Indian-ancestry rhesus macaques between 2 to 23 years old and found that aging negatively correlated with neutrophil counts and positively correlated with plasma G-CSF levels. Hierarchical clustering analysis suggested that G-CSF was strongly associated with pro-inflammatory cytokines IL-1b and MIP-1a. In elderly rhesus macaques, neutrophils expressed less myeloperoxidase and there was a higher frequency of PMN-MDSCs compared to the young adult macaques. In summary, there appears to be a dysregulated feedback mechanism wherein increased levels of G-CSF failed to restore neutrophil production in elderly rhesus macaques that was associated with induced production of pro-inflammatory cytokines and earlier release of less mature neutrophils and PMN-MDSCs. Together, these findings may contribute to the understanding of the chronic inflammation and greater susceptibility to infectious diseases in the elderly.
Baby’s First Macrophage: How do placental macrophages (Hofbauer Cells, HCs) contribute to immune tolerance and infection response during pregnancy?
Authors: Swieboda Dominika , Johnson Erica , Skountzou Ioanna , Chakraborty Rana
Published: January 2023

Placental immunity is dichotomous: tolerance of the semiallogenic fetus is balanced with limiting transmission of maternal pathogens. HCs are the major fetal immune cell at the placenta, but mechanisms responsible for maintaining immune homeostasis while preventing infection require elucidation. We determined the phenotype of human HCs throughout gestation and analyzed stimulation response.

Activated HCs were present in early pregnancy and reduced in number by term while maintaining similar phenotypes. Tolerant HC numbers were highest in midgestation, after a relatively intolerant phenotype early in gestation. We saw a significant shift in macrophage polarization as gestation progressed. Transcription of ARG2 exceeded iNOS at all points, reaching 10-fold higher at term.

Following treatment with IL4+IL13 or IFNγ+LPS midgestation HCs underwent significant phenotype change and activation. Basal expression of antiviral IFN stimulated genes (ISGs) was lowest at midgestation and was enhanced by IFN-α and IFN-λ1 with a 10-fold stronger response to IFN-α. RIG-I agonism induced HC activation, 10-fold iNOS upregulation and enhanced transcription of IFNs, MDA5, RIG-I, and ISGs.

Response to stimulation by IFNγ+LPS, IL4+IL13, IL1β+HAGG was limited to loss of tolerance at term. IL-10 treatment increased numbers of CD163+ HCs, and IFNγ+LPS caused loss of discernible polarization patterns. Basal expression of RIG-I, MDA5 and ISGs was highest in term HCs, but IFN-α did not activate them. RIG-I agonism reduced markers of tolerance.

HCs are variable macrophages, with phenotype and immune capacity strongly dependent on gestational age. Understanding placental immunobiology is fundamental to addressing key pregnancy morbidities.
Distribution and phenotypes of SLAMF9+ myeloid cells in mouse tissues
Authors: Mikulin Joseph A , Wilson Timothy J
Published: January 2023

SLAM family receptors are critical mediators of immune function, whose expression varies across tissue and cell type. However, the expression of SLAM Family Member 9 (SLAMF9), the most recently discovered protein in the family, has yet to be investigated in mouse or human tissues. In this study, we sought to discover in what tissues and the degree to which SLAMF9 is expressed in the mouse model, as well as determine whether the cells expressing SLAMF9 belong to either a known or a novel subset. We discovered that, within the non-classical monocyte subset (CD45+CD11b+Ly6c-), two broad groups emerged based on SLAMF9 expression. The SLAMF9+ cells in this subset appear to constitute a homogenous group in both the kidney and liver. Renal SLAMF9+ cells also demonstrate higher expression of MHC-II, CX3CR1, and CD11c than their SLAMF9-counterparts, as well as low F4/80 expression and no CD103 expression. However, a cell type with largely similar marker expression and lower frequency appears in the hepatic non-classical monocyte gate, indicating that the cell type may be conserved across tissues. Therefore, SLAMF9 marks a subset of CD103−, CD172a+ classical dendritic cells and may designate a specialized subset of these cells with similar functions in both the liver and kidneys.
Maternal EGF limits bacterial translocation in the offspring: a model of enterally acquired Late Onset Sepsis
Authors: Knoop Kathryn A , Coughlin Paige , Floyd Alexandria , Hall-Moore Carla , Ndao Malick , Warner Barbara , Tarr Phillip , Newberry Rodney
Published: January 2023

Late-onset neonatal sepsis (LOS), an important cause of morbidity and mortality in prematurely born infants, results from a bloodstream infection by bacteria of gut origin. It remains unclear how such bacteria translocate from the intestine, though it has been repeatedly observed that breastfed infants, particularly those fed mother’s own milk (MOM), have reduced risk of LOS compared to formula fed infants. Epidermal growth factor (EGF) promotes intestinal barrier function in infants and is present at high concentrations in breast milk post-partum and decreases throughout lactation. We found reduced concentrations of EGF in the stool of premature formula-fed infants compared to MOM-fed infants, and observed a similar decrease in EGF concentrations of stool of neonatal mice asynchronously cross-fostered (ACF) to dams that had delivered two weeks prior. LOS bloodstream isolates of E. coli colonized the tracts of all pups but translocated and disseminated systemically only in ACF mice resulting in bacteremia and rapid death. Oral gavage of recombinant EGF reduced bacteria translocation and prevented the development of systemic disease in ACF mice. Thus, disruption of maternally delivered EGF in ACF mice results in translocation of pathogens from the gut, and a sepsis-like disease. In conclusion we have identified a mechanism whereby gut-residing pathogens gain systemic access and have developed a novel animal model replicating this mechanism to explore the protective effect of breastmilk and EGF in LOS.
PTP1B negatively regulates Pseudomonas aeruginosa killing by neutrophil through TLR4-STAT1-iNOS signaling
Authors: Yue Lei , Yan Min , Li Hua , Yang Ting , Lin Tong-Jun , Xie Zhongping
Published: January 2023

Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals and chronic obstructive pulmonary diseases (COPD) patients. Mechanisms involved in immune responses to P. aeruginosa infection and bacterial clearance remain incompletely defined. Here we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator in P. aeruginosa killing by neutrophil. PTP1B-deficient neutrophil display greatly enhanced bacterial killing capability and TLR4 transcription following P. aeruginosa infection. The negative regulation of PTP1B on the production of nitric oxide by neutrophil is blocked by TLR4 antagonist. Furthermore, the STAT1 activation in neutrophils following P. aeruginosa infection occurs in the downstream of TLR4, and PTP1B deficiency leads to enhanced TLR4 dependent STAT1 activation and iNOS expression by neutrophil. Interestingly, PTP1B deficiency mainly up-regulates the production of pro-inflammatory cytokines by neutrophil, including IL-6, IL-1β and TNFα, but only IL-6 is blocked by TLR4 antagonist. Further studies reveal that PTP1B and STAT1 are physically associated. These findings demonstrate a novel regulatory mechanism of the immune response to P. aeruginosa infection through PTP1B-STAT1 interaction. This novel PTP1B-STAT1-iNOS pathway may have broader implications in Toll-like receptor mediated innate immunity.
Expression of human TLR4/MD-2 directs an early innate immune response associated with modest increase in bacterial burden during Coxiella burnetii infection
Authors: Hedges Jodi F , Robison Amanda , Snyder Deann Teresa , Hajjar Adeline M. , Jutila Mark A
Published: January 2023

Human and mouse TLR4 molecules respond differently to hypo-acylated LPS. The LPS of Coxiella burnetii is hypo-acylated, heavily glycosylated and known to cause a minimal response by human cells. We hypothesized that mice expressing hTLR4 molecules would be more susceptible to C. burnetii infection. Our results suggested that transgenic mice expressing hTLR4 and the human MD-2 adaptor protein (hTLR4/MD-2) generally respond similarly to wild type mice, contrary to our hypothesis. Small differences in bacterial burdens in spleens and/or lungs of infected mice were noted and lung pathology was increased in hTLR4/MD2 compared to wild type mice. Surprisingly, bone marrow chimera experiments indicated that hTLR4/MD-2 expression on non-hematopoietic cells, rather than the target cells for C. burnetii infection, accounted for increased bacterial burden. Very early during infection, cytokines involved in myeloid cell recruitment were detected in the plasma of hTLR4/MD2 mice, but not in wild type mice. This restricted cytokine response was accompanied by early neutrophil recruitment to the lung in hTLR4/MD2 mice. These data suggest that hTLR4/MD-2 may alter early responses to C. burnetii infection. Our results suggest that during C. burnetii infection, the species of origin for TLR4 may be less important than with other pathogens with hypoacylated LPS.
Innate immune role for the Macrophage Galactose Lectin (MGL) pathway in pulmonary tuberculosis
Authors: Naqvi Kubra Fatima , Saito Tais B , Huante Matthew B , Gelman Benjamin B , Endsley Janice J
Published: January 2023

Pulmonary tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (Mtb), remains a significant public health threat and accounts for 1.3 million deaths each year. Innate immune responses to Mtb through pathogen recognition receptor signaling are capable of producing protective immune responses and controlling infection. To date, however, an anti-mycobacterial role for the C-type lectin receptor Macrophage Galactose Lectin (MGL), present on the surface of alternatively activated macrophages and dendritic cells, has not been described. MGL has been shown to be an important anti-inflammatory immune receptor during Klebsiella pneumoniae pneumonic sepsis, suggesting its involvement during lung infections; although the role of MGL during TB has not been studied. In preliminary studies, we identified HIV-mediated disruption of MGL gene expression in the lungs of humanized mice co-infected with HIV and Mtb by using a custom macrophage transcriptome array. To further investigate a role for how loss of MGL activation would affect innate immunity to Mtb, we compared disease following pulmonary challenge of MGL−\− and WT mice with Mtb H37Rv. Compared to WT mice, we observed greater mycobacterial burden in the lungs of MGL−\− mice. We additionally observed differences in pulmonary pathology including the accumulation of foamy macrophages and altered patterns of leukocyte organization in granulomas. These data suggest that MGL signaling may regulate myeloid cell antimicrobial activity and inflammatory outcomes at sites of Mtb infection in the lung.
Egr-1 deficiency protects host against Pseudomonas aeruginosa lung infection
Authors: Pang Zheng , Raudonis Renee , Cheng Zhenyu
Published: January 2023

Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of nosocomial infections in cystic fibrosis patients and immunocompromised individuals. The molecular mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Early Growth Response 1 (Egr-1) is a zinc-finger transcription factor that binds to the GC-rich DNA consensus sequences in the promotor of target genes, important for cell growth, differentiation and survive. Aberrant expression of Egr-1 has been implicated in many inflammatory diseases. In this study, we demonstrate that Egr-1 deficiency protects host against P. aeruginosa infection in a mouse model of acute bacterial pneumonia. Egr-1 expression was rapidly and transiently induced both in vitro and in vivo upon P. aeruginosa infection. Egr-1-deficient mice displayed decreased disease score, reduced systemic levels of proinflammatory cytokines and impaired NF-κB and NFAT activation compared to wild-type mice. Interestingly, Egr-1 deficiency leads to enhanced bacterial clearance and increased nitric oxide production in lung whereas it has no impact on neutrophil recruitment. Further studies revealed that Egr-1-deficient neutrophils displayed elevated bacterial killing ability. Altogether, these findings suggest that Egr-1 promotes inflammatory responses by enhancing NF-κB and NFAT activation and plays a detrimental role in host defense against P. aeruginosa lung infection by negatively regulating nitric oxide production for bacterial clearance.
Suppression of human STAT2 by ZIKV NS5
Authors: Hai Rong , Wang Boxiao , Thurmond Stephanie , Song Jikui
Published: January 2023

The infection of ZIKA virus (ZIKV), a mosquito-transmitted flavivirus, has been shown to cause congenital disease in the context of infection during pregnancy in its 2015 outbreak. To establish the infection, ZIKV must suppress host interferon (IFN) response. Previous studies have shown that ZIKV utilizes its non-structural NS5 protein to trigger the human STAT2, a critical mediator to boost host interferon (IFN) response, for the proteasome-dependent degradation. However, the underlying mechanisms of viral NS5 and hSTAT2 remain elusive largely due to the lack of atom level conformational information. Here, we report the structure of hSTAT2 in complex with ZIKV NS5. hSTAT2, with the SH2 domain occluded by the C-terminal tyrosine-phosphorylation (pY)-tail segment, inserts the coiled-coil domain into the cleft between the methyltransferase and RdRP domains of NS5 in a manner that would occlude the association of hSTAT2 with cellular regulatory protein IRF9. Disruption of the ZIKV NS5 – hSTAT2 association in cells compromises the NS5-mediated hSTAT2 degradation and IFN responses. This study provides a basis for understanding the functional antagonism between viral antagonists and STAT2.
Dual oxidase1 ameliorates survival, viral clearance and pulmonary pathology during influenza infection
Authors: Sarr Demba , Gingerich Aaron , Colon Maria , Nagy Tamas , Tripp Ralph A , Rada Balazs
Published: January 2023

Influenza virus infections cause severe morbidity and mortality, especially in high-risk patient populations. Bronchoepithelial cells orchestrate an oxidative antimicrobial system present in the airway liquid consisting of lactoperoxidase, the thiocyanate ion, and hydrogen peroxide. Dual Oxidase 1 (Duox1), an enzyme highly expressed in these cells, is the source of hydrogen peroxide. While in vitro studies suggest an anti-influenza, and a general antimicrobial role of Duox1, its antiviral role in vivo has not been addressed so far. We hypothesized that Duox1 has a protective effect in vivo against influenza. To test this hypothesis, we infected wild-type C57BL/6 and Duox1-deficient mice intranasally with the mouse-adapted A/Puerto Rico/8/1934 H1N1 (PR8) influenza virus strain. To evaluate the clinical and immunological significance of Duox1 in influenza infection, multicolor flow cytometry, multiplex ELISA, viral titration, histology and immunostaining techniques were used. Our results indicate that Duox1-deficient mice have increased mortality following influenza infection. Weight loss of Duox1-deficient mice post-infection was also significantly higher than in Duox1-expressing animals. Duox1 also limited influenza virus replication in the lung. Characterization of infiltrated leukocyte subsets following influenza infection revealed impaired natural killer cell recruitment in Duox1-deficient animals while other lymphoid or myeloid leukocyte subsets were not affected In summary, our results provide the first evidence for the in vivo antiviral role of Duox1, and also implicate that targeting the Duox1-based system has the potential to provide novel treatment or prophylactic strategies against influenza.
Dysregulation of innate interferon signaling by Epstein-Barr virus tegument protein BGLF2
Authors: SONIA  , Yuen Kit-San , Chaudhary Vidyanath , Botelho Michael George , Jin Dong-Yan
Published: January 2023

Epstein-Barr virus (EBV) successfully infects more than 90% of adults, causing lymphoid or epithelial malignancies such as lymphomas, nasopharyngeal and gastric carcinomas only in a small subset of infected people. EBV can switch between latency and lytic replication in B lymphocytes and epithelial cells. To maintain its life cycle, EBV has to develop mechanisms to evade host innate immune responses. In this study we identified EBV tegument protein BGLF2 as another viral modulator of JAK-STAT-dependent innate immune responses. BGLF2 interacted with STAT2 and promoted its degradation by enhancing K48-linked ubiquitination. BGLF2 also inhibited phosphorylation of JAK1 and STAT1, probably by recruiting a tyrosine phosphatase to these targets. As a result, BGLF2 suppressed ISRE-dependent transcription of interferon (IFN)-stimulated genes (ISGs) induced by type I IFNs. In addition, BGLF2 also exerted an inhibitory effect on type II and type III IFN signaling. Pre-treatment of host cells with IFN-β desensitized them for EBV primary infection and reactivation. Expression of BGLF2 in IFN-β-pre-treated cells reversed IFN-β-mediated inhibition and restored EBV infectivity. Thus, BGLF2 might subserve an IFN-antagonizing function in EBV lytic infection. Our findings reveal a novel EBV modulator of innate IFN response. Supported by HMRF 17160822, RGC C7027-16G and AoE/M-06/08.
Transient Receptor Potential Vanilloid 4 (TRPV4) Regulates the Host Defense Response to Bacterial Pneumonia Through a MAPK Molecular Switch
Authors: Scheraga Rachel Greenberg , Abraham Susamma , Grove Lisa , Southern Brian D , Crish James , Hamilton Thomas A , McDonald Christine , Olman Mitchell
Published: January 2023

We have shown that macrophage activation in response to lipopolysaccharide (LPS) is sensitive to the biophysical properties of the matrix through the mechanosensitive channel, transient receptor potential vanilloid 4 (TRPV4). However, the signals that drive macrophage activation are not fully known. Studies suggest that LPS-induced macrophage activation is controlled in part by the MAPK pathway (i.e. p38, ERK, JNK). Thus, we investigated if TRPV4 mediates the macrophage response to LPS through MAPKs. In TRPV4 deleted BMDMs, p38 activation was decreased (69%) while JNK activation was increased (2-fold), and ERK was unchanged after LPS, in a matrix stiffness-dependent manner. Further studies reveal that activation of p38 enhances LPS-induced macrophage phagocytosis (2-fold), but blocks IL-1β secretion (2-fold); while JNK enhances secretion of IL-6, IL-8, and MIP-2 (2 fold). Hence, the TRPV4 and LPS signals synergizes to activate p38 which increases phagocytosis and limits IL-1β secretion while downregulating JNK activation to limit other pro-inflammatory cytokines. In order to evaluate the role of TRPV4 in chronic pneumonia/lung injury, WT and TRPV4 KO mice were intratracheally administered agarose-embedded P. aeruginosa. Loss of TRPV4 led to decreased phagocytosis/bacterial clearance (6-fold), increased lung inflammation as measured by inflammatory cell infiltration (80±3%), vascular permeability (63±6%), and pro-inflammatory cytokine secretion (IL-1β > by 71±4%). Thus, in the setting of lung infection TRPV4 functions to induce a molecular switch towards p38 which mediates bacterial clearance and downregulates pro-inflammatory cytokine secretion, to reduce infection-associated lung inflammation.
BATF-interacting proteins dictate specificity in Th subset activity
Authors: Fu Yongyao , koh Byunghee , Ulrich Benjamin J. , Kharwadkar Rakshin , Kaplan Mark H.
Published: January 2023

The basic leucine zipper transcription factor BATF is expressed in multiple Th subsets and cooperates with other factors to regulate gene transcription. BATF activates lineage-specific cytokines in Th subsets, activating IL-9 in Th9 cells and IL-17 in Th17 cells. The mechanism for this restricted activity is unclear. In this report we define BATF binding partners that contribute to Th subset-specific functions. Although BATF and IRF4 are expressed in greater amounts in Th9 than Th17, increased expression of both factors is not sufficient to induce IL-9 in Th17 cells. BATF also requires heterodimer formation with Jun family members to bind DNA and induce gene expression. We observed that JunB and c-Jun, but not JunD, promote IL-9 production in Th9 cells. Ectopic expression of BATF with either JunB or c-Jun generates modest but significant increases in IL-9 production in Th17 cells, suggesting that the low expression of Jun family members is one factor limiting the ability of BATF to induce IL-9 in Th17 cells. We further identified that Bach2 positively regulates IL-9 production by directly binding to the Il9 gene and by increasing transcription factor expression in Th9 cells. Strikingly, co-transduction of Bach2 and BATF significantly induces IL-9 production in both Th9 and Th17 cell. Taken together, our results reveal that JunB, c-Jun and Bach2 cooperate with BATF to contribute to the specificity of BATF-dependent cytokine induction in Th subsets.
Cold . . . is not so cold: CD4+ T cells are active but suppressed in colorectal cancer
Authors: Jessup J. Milburn
Published: January 2023

Introduction

Checkpoint inhibitors blocking the PD-1/PD-L1 axis or CTLA4 are standard of care for lung and kidney cancer and melanoma. However, colorectal carcinoma (CRC), the second most lethal cancer in the US, has only a 7% response rate to checkpoint inhibitors because more than 60% of CRC lack tumor infiltrating lymphocytes, PD-L1 expression, or have low mutational burden. We postulated that cellular immune responses to autologous tumor antigens in patients with CRC are present but suppressed.

Methods

Patients with stage I – III CRC were studied as part of a vaccine program to determine cellular immunity to their autologous tumor. Single cell suspensions of tumor and normal mucosa were made and cryopreserved from each patient. All patients had IgG or IgA antibodies that reacted with autologous tumor by indirect immunofluorescence. Peripheral blood mononuclear cells (PBMC) were isolated and CD4+ and CD8+ T cell subsets were cultured with Mitomycin C-treated autologous Tumor or mucosal cells. Proliferation was measured after 3 days by 3H-thymidine post-labeling.

Results

4 of 5 unvaccinated patients had CD4+ T cell subset proliferation whereas PBMC or CD8+ T cell subsets did not. When CD8+ T cells were added back to CD4+ T cells, proliferation was inhibited. Patients vaccinated with BCG and autologous tumor cells displayed specific cellular-immune responses for up to 9 months.

Conclusion

CRC patients with T-dependent IgG or IgA autoantibodies frequently contain a CD4+ T cell subset that may be boosted to support systemic immunity or, possibly, checkpoint inhibitor therapy.
Peroxynitrite affects MHC I peptide repertoire presented on tumor cells and impedes the efficacy of antitumor cytotoxic T-lymphocytes
Authors: Tcyganov Evgenii , Gabrilovich Dmitry I
Published: January 2023

Despite the advancements in enhancing antigen-specific T cell responses in cancer their clinical benefit remains limited. Recent studies demonstrated that tumor microenvironment factors are able to dampen in vivo anti-tumor T cell response. Previously, we found that myeloid cells produce large amounts of peroxynitrite (PNT) in the tumor. The pretreatment with PNT blocked binding of exogeneous peptides by tumor MHC I and decreased tumor killing by cytotoxic T lymphocytes (CTL). We hypothesized that PNT affects tumor MHC I peptide repertoire, resulting in inability of CTL to recognize and eliminate tumor cells. To test this hypothesis, we treated tumor cells with PNT and analyzed MHC I peptides by LC-MS/MS. We found that PNT dramatically affected the repertoire of tumor MHC I peptides. We identified two groups of peptides: “stable” peptides that were retained on MHC class I after PNT treatment and “non-stable” peptides that were significantly reduced after treatment. The characterization of peptides had revealed similar binding capacity for both “stable” and “non-stable” peptides. However, the dissociation rate of “non-stable” peptides from peptide-MHC I complexes was significantly higher. We found that PNT treatment impaired proteasomal activity in tumor cells. This may skew MHC I repertoire towards more “stable” peptides. To examine biological relevance of PNT, we generated CTLs specific to representative “stable” and “non-stable” peptides. We confirmed that PNT pretreatment of tumor cells decreased the killing efficacy of CTLs specific to “non-stable”, but not to “stable” peptides.

Overall, our findings demonstrate a novel PNT-mediated mechanism of tumor cell resistance to CTLs that could be targeted for cancer immunotherapy.
Suboptimal ER stress Induced Autophagy Potentiates Anti-tumor T Cell Response
Authors: Chakraborty Paramita , Chatterjee Shilpak , Tran Danh , Nadig Satish , Atkinson Carl , Mehrotra Shikhar
Published: January 2023

Endoplasmic reticulum (ER) stress induced by external or internal stimuli, activates a number of well-orchestrated cellular signaling processes aiming either to promote cell apoptosis or to restore cellular function and resolve the stress. In tumor microenvironment, induction of ER stress is known to dampen the antitumor activity of T cells by reducing their mitochondrial function. However, if magnitude of ER stress governs the T cell fate and function is unknown. Using melanoma antigen gp100 reactive T cells, we found that low level of ER stress enhances T cell stemness and promotes mitochondrial biogenesis, whereas high level of ER stress triggers T cell death. Moreover, upon adoptive transfer, T cells treated with low dose ER stress inducer are able to form long-lived memory in vivo, express reduced level of co-inhibitory molecule, and demonstrate superior anti-tumor immunity by increasing overall survival of B16 melanoma bearing mouse. Mechanistically, we discovered that, upon ER insult at suboptimal level, a protective autophagy pathway is induced to promote cell survival and maintain stemness through the protein kinase R-like endoplasmic reticulum kinase (PERK)/ activating transcription factor-4 (ATF4)-dependent manner. Conversely, knockdown of PERK abrogates autophagy activation, hampers mitochondrial biogenesis in response to suboptimal ER stress, which in-turn compromises the antitumor function of melanoma antigen specific T cells. Overall, these preclinical data highlights that, low level of ER stress is important for healthy cellular function and therapeutically, ER stress pathways can be manipulated in T cells in order to regulate their antitumor potential.