bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of NF‐kB1 by OX40 contributes to antigen‐driven T cell expansion and survival
Authors: Song Jianxun , So Takanori , Croft Michael
Published: June 2021

The costimulatory molecule OX40 (CD134) is strongly required in many instances for effective T cell‐mediated immunity, controlling proliferation and survival of T cells after encountering specific antigen. We previously found that the functional targets of OX40 are survivin and aurora B that regulate proliferation, and Bcl‐2 anti‐apoptotic family members that regulate survival. However, the intracellular pathways from OX40 that mediate these effects are unclear. Here, we show that OX40 signaling can target the canonical NF‐kB (NF‐kB1) pathway in peripheral antigen‐responding CD4 T cells. Phosphorylation of IkBalpha which leads to NF‐kB1 (p50)/RelA nuclear translocation is impaired in OX40‐deficient T cells. Retroviral transduction of active IKKbeta that constitutively activates NF‐kB1 rescues the poor proliferation and survival of OX40‐deficient T cells, directly correlating with increased expression and activity of survivin, aurora B, and Bcl‐2 family members. Moreover, active IKKbeta expression alone is sufficient to restore the defective proliferation and survival of OX40‐deficient T cells in vivo when responding to antigen. Thus, OX40 signals regulate T cell number and viability through the NF‐kB1 pathway that controls expression and activity of intracellular targets for proliferation and survival.
B7‐DC‐expressing Dendritic Cells Sequentially Promote and Limit Th1, Th2, and Th17 Responses in Lung‐draining Lymph Nodes
Authors: Oriss Timothy B. , Krishnamoorthy Nandini , Dixon‐McCarthy Barbara , Arora Meenakshi , Zheng Mingquan , Kolls Jay K. , Ray Prabir , Ray Anuradha
Published: June 2021

Under conditions that result in inflammation of the airways, increased expression of the “non‐conventional” co‐stimulatory molecule B7‐DC was observed on myeloid‐like dendritic cells (mDCs) in the lung‐draining lymph nodes. This enhanced B7‐DC expression was observed in murine models of airway inflammation which result in skewing towards Th1‐, Th2‐, and Th17‐biased responses and were generated using administration of OVA plus CpG ODN, OVA plus cholera toxin, or infection with Klebsiella pneumoniae, respectively. B7‐DC expression on mDCs early in the immune response was associated with enhanced T cell cytokine production whereas later in the immune response, mDCs expressing B7‐DC were less stimulatory than those lacking its expression. Inhibitory effects were at least partially mediated via T cell expression of PD‐1, the inhibitory ligand for B7‐DC, and stimulatory effects were assumed to occur through the presently unidentified co‐stimulatory ligand which has been proposed for B7‐DC. The results indicate that under Th1, Th2, as well as Th17 conditions, B7‐DC expression appears to promote T cell cytokine production early, but appears to participate in limiting chronic activation of cells in later stages of the immune response. This work was supported by NIH grants HL77430 to A.R., HL60207 to P.R., and an American Lung Association Dalsemer Award to T.O.
PD‐L1 interaction limits excessive Th1 effector function during Mycobacterium tuberculosis infection
Authors: Barber Daniel L. , Antonelli Lis R. , Feng Carl G. , Sharpe Arlene H. , Freeman Gordon J , Sher Alan
Published: June 2021

Mycobacterium tuberculosis establishes a persistent infection despite a T cell response that contains but does not clear the pathogen. The B7 family member PDL1 has been shown to play a major role in regulating T cell responses during chronic viral infection. Here we investigate its role in regulating T cell function during murine Mtb infection. PD‐1 was upregulated on T cells in lung and spleen after low‐dose aerosol infection and PDL1 was markedly increased on multiple cell types. αPDL1 administration from wk10–14 had no effect on bacterial control but led to a 2–3 fold reduction in the number of IFNγ+ T cells. Furthermore, in contrast to previous reports with virus specific T cells, PDL1 blockade during culture of splenocytes from Mtb infected mice reduced T cell proliferation and triggered enhanced apoptosis. In the presence of αIFNγ neutralizing mAb or aminoguanidine, however, in vitro PDL1 blockade enhanced CD4 T cell division suggesting that this pathway protects T cells from suppression by IFNγ and nitric oxide by limiting their production. Moreover in recent experiments Mtb infected PDL1 KO mice were found to display acute mortality succumbing by day 31 post‐exposure. Taken together these data suggest that a major function of PDL1 in Mtb infection may be to limit potentially host detrimental Th1 responses.
Peroxiredoxin II Regulates CD8+ T Cell Expansion and Survival During Acute and Chronic Viral Infections
Authors: Michalek Ryan D , Weant Ashley E , Moon Eun‐Yi , Yu Dae‐Yeul , Grayson Jason M
Published: June 2021

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. We examined the role of the antioxidant enzyme, Peroxiredoxin II (PrxII), during T cell activation and throughout the immune response to acute and chronic viral infections. Following TCR stimulation, PrxII levels increased 50‐fold by 30 hours. Examination of differentiated T cells demonstrated that effector cells possessed lower PrxII levels than naïve or memory CD8+ T cells. Utilizing mice deficient in PrxII, we determined that ROI levels measured in T cells, either directly ex vivo or in response to stimulation, were higher than wild‐type mice. Deletion of PrxII also increased in vitro T cell proliferation, but decreased cell survival during division. Acute infection of PrxII−/− mice with LCMV Armstrong revealed an increased expansion of effector CD8+ T cells on day 8, but yielded no significant difference in memory CD8+ T cells by day 97. Additionally, no differences were observed in virus‐specific CD4+ T cells or antibody secreting cells at any time during infection. In contrast, chronic infection with LCMV Clone 13 resulted in death by day 13 post infection. Together, these results demonstrate that redox regulation of T cell activation through PrxII is critical during acute and chronic viral infections.

Supported by ACS‐ #RSG‐04‐066‐01‐MBC to J.G.
TWEAK differentially regulates the production of inflammatory mediators from fibroblast‐like synoviocytes of rheumatoid arthritis
Authors: Kang Young Mo , Sa Keum Hee , Kim Ji Min , Han Seung Woo , Lee Won Ha , Nam Eon Jeong
Published: June 2021

Objectives: To investigate the regulatory role of TWEAK on production of effector molecules from fibroblast‐like synoviocytes (FLS) in rheumatoid arthritis (RA).Methods: The expression of TWEAK and Fn14 was evaluated using immunohistochemical study. After stimulation of FLS with TWEAK, gene expression microarray assay was performed. The expression of Fn14 and effector molecules was determined by RT‐PCR, Western blot analysis, and flow cytometry. Results: The expression of TWEAK and Fn14 was up‐regulated within synovial tissues of RA. The expression of Fn14 on the FLS was induced by PMA but not by FGF‐acidic or ‐basic. When the expression gene profile was evaluated, transcripts of MMPs were down‐regulated with TWEAK treatment. To confirm the results, FLS was stimulated with soluble TWEAK (100ng/ml), which increased ICAM‐1 and VCAM‐1, but down‐regulated MMP‐1 and TIMP‐1 slightly. TWEAK showed an additive effect on IL‐1¥â‐ and IFN‐¥ã‐induced up‐regulation of ICAM‐1 & VCAM‐1, but no additional effects on the TNF‐¥á‐induced up‐regulation. TWEAK significantly inhibited IL‐1¥â‐induced production of MMP‐1 and TIMP‐1. Although COX‐2 expression was highly upregulated by IL‐1¥â, Tweak did not make any additional changes. Conclusions: Our data showed that the interaction between TWEAK and Fn14 may involve in the pathogenesis of RA by differentially regulating the synthesis of effector molecules from FLS.
TGF‐beta enhances the laminin‐1‐induced production of IL‐16 in RA synovial fibroblasts by elevation of beta1‐integrin expression
Authors: Warstat Katrin , Tsui Shanli , Pap Thomas , Essl Mike , Smith Terry J , Cruikshank William W , Klein Gerd , Gay Steffen , Aicher Wilhelm K.
Published: June 2021

Objectives

Synovial tissue of rheumatoid arthritis (RA) patients is characterized by strong expression of laminins and release of inflammatory cytokines, one of which is IL‐16. In this study we investigated regulatory pathways of IL‐16 in synovial fibroblasts (SF) from RA and osteoarthritis (OA) patients.

Methods

We analyzed attachment to LM‐111 and expression of integrins in SF. Expression of IL‐16 in SF grown on LM‐111 w/o TGF‐beta was quantified by qRT‐PCR and ELISA. Activity of IL‐16 was monitored in a cell migration assay. TGF‐induced signaling pathways were studied by immunoblotting and by blocking with p38MAPK‐, MEK‐, and Smad2 inhibitors.

Results

TGF‐activated SF showed an upregulation of beta1‐integrin and adhered more closely to LM‐111. This attachment to LM‐111 could be blocked by mAb to beta1‐integrin. Stimulation with TGF and growth on LM‐111 significantly increased IL‐16 mRNA and protein in RA‐SF. In OA‐SF, TGF failed to induce production of IL‐16. Expression of IL‐16 was regulated by p38MAPK, MEK‐ERK1/2 and Smad2 signaling.

Conclusion

The adherence of RA‐SF to LM‐111 in the presence of TGF triggers a significant IL‐16 response and thus contributes to the infiltration of CD4+ lymphocytes into synovial tissues. This mode of induction of IL‐16 represents a novel pathway that results in IL‐16 production in RA‐SF but not in OA‐SF.
Intracellular accumulation of mutant TNFR1 potentiates MAP‐Kinase signaling and innate immune responses in the TNF‐receptor associated Periodic Syndrome (TRAPS)
Authors: Park Heiyoung , Simon Anna , Maddipati Ravikanth , Bulua Ariel , Komarow Hirsh , Kastner Daniel , Siegel Richard M
Published: June 2021

Tumor necrosis factor (TNF) receptor‐associated periodic syndrome (TRAPS) is an autosomal dominant auto‐inflammatory syndrome associated with mutations in the gene that encodes tumor necrosis factor receptor 1 (TNFR1). How these mutations predispose to inflammation is not known. We have previously shown that TRAPS‐related mutant TNFR1 failed to bind TNF or traffic to the cell surface. These mutants exhibited abnormal oligomerization and are retained in the endoplasmic reticulum (ER). In both knock‐in mice and patient PBMC, TRAPS mutant TNFR1 accumulates intracellularly and has a prolonged half‐life. This suggests that intracellular accumulation of mutant TNFR1 is linked to disease in TRAPS. Although cells harboring TRAPS mutant TNFR1 were defective in signaling in response to TNF, MAP kinases were found to be constitutively activated, and stimulation with LPS resulted in prolonged activation of JNK, ERK and p38 kinases with intact NF‐κB activation. These signaling abnormalities led to dramatically increased TNF‐independent production of IL‐6 in cells with one or both mutant TNFR1 alleles. Increased IL‐6 production was dependent on Reactive Oxygen Species, which have been previously been shown to sustain MAPK signaling after TNF treatment. We conclude that intracellular mutant TNFR1 in TRAPS potentiates innate immune signaling through abnormal activation of MAPK and ROS.
Neuropilin‐1 is a receptor for latent and active TGFβ‐1and is involved in suppression by regulatory T cells
Authors: Glinka Yelena , Prud’homme Gerald J.
Published: June 2021

Neuropilin‐1 (Nrp1) is a receptor for the class 3 semaphorins and vascular endothelial growth factor (VEGF). It is also a marker of regulatory T cells (Tr), which often carry both Nrp1 and latency‐associated peptide‐TGFβ‐1. The signaling TGFβ‐1 receptors bind only active TGFβ‐1, and we hypothesized that Nrp1 binds the latent form.

Indeed, we found that Nrp1 is a high affinity receptor for both latent and active TGFβ‐1. Free LAP and LAP‐TGFβ‐1 competed with VEGF165 for binding to Nrp1. LAP has a basic, arginine‐rich C‐terminal motif similar to VEGF and peptides, which bind to the b1 domain of Nrp1. A C‐terminal LAP peptide (QSSRHRR) bound to Nrp1 and inhibited the binding of VEGF and LAP‐TGFβ‐1.

Compared to Nrp1‐ cells, sorted Nrp1+ T cells had a much greater capacity to capture LAP‐TGFβ‐1. Sorted Nrp1‐ T cells captured soluble Nrp1‐Fc, and this increased their ability to capture LAP‐TGFβ‐1.

Conventional CD4+CD25‐Nrp1‐ T cells double‐coated with Nrp1‐Fc/LAP‐TGFβ‐1 acquired strong Tr activity. Moreover, LAP‐TGFβ‐1 was activated by Nrp1‐Fc, and also by a peptide of the b2 domain of Nrp1 (RKFK; similar to a thrombospondin‐1 KRFK peptide, which is involved in LAP‐TGFβ‐1 activation). Nrp1 also activated LAP‐TGFβ‐1 in cell‐free system.

Thus, Nrp1 is a receptor for latent and active TGFβ‐1, and can contribute to Tr activity.
Differential beta‐3 integrin of CXCR3, CXCR4, and CXCR7 clustering on breast cancer cells in response to CXCL11, CXCL12, and Adrenomedullin
Authors: Mosley LaSharon Denise , Sharma Praveen K , Singh Rajesh , Singh Shailesh , Lillard James W
Published: June 2021

Metastasis is responsible for essentially all breast cancer (BrCa) related deaths; therefore, therapies designed to prevent the spread of BrCa cells are greatly needed. Similarly, the anti‐apoptotic mechanisms employed by BrCa cells to survive in serum‐free environments and apoptosis‐inducing therapeutics are numerous and contribute significantly to the morbidity and mortality of this disease. Chemokines have been demonstrated to support BrCa metastasis and may also activate anti‐apopototic mechanisms. In this regard, BrCa cells express functionally active CXCR3, CXCR4, and CXCR7 to presumably support adhesion, invasion, and cell survival. The ligands for CXCR7 are CXCL11 (also binds CXCR3), CXCL12 (also binds CXCR4), calcitonin‐related gene peptide (CRGP), and adrenomedullin (AM). We provide evidence that CXCR7 expression correlates with breast tumor staging and confirm that this receptor; along with CXCR3 and CXCR4, are expressed by BrCa cell lines. We also show that CXCL11, CXCL12, and AM, but not CRGP, differentially modulates β3 integrin clustering and CXCR3, CXCR4, and CXCR7 aggregation in BrCa cell lines. Chemokine receptor aggregation coupled to integrin clustering induces signals to support cell survival and adhesion. Taken together these studies provide important and novel information regarding the cellular and molecular mechanisms of CXCR3, CXCR4 and CXCR7 in breast tumor progression.
The acquisition of narrow binding specificity by polyspecific natural IgM antibodies in a semi‐physiological environment
Authors: Sokoloff Alex V , Chu Qili , Ludtke James J , Subbotin Vladimir M , Blochin Andrey
Published: June 2021

Natural IgM Abs play an important role in clearing pathogens and preventing autoimmunity. The molecular mechanisms that mediate these processes are understood only to a limited degree. This shortcoming is largely due to the fact that isolated natural IgM Abs commonly recognize a variety of unrelated antigens (Ags). Such polyspecificity is generally believed to be an inherent property of natural Abs. However, there is increasing evidence that it may be induced by non‐physiological binding conditions. In this study, we compared the specificity of natural IgM Abs in conventional buffers and undiluted sera deficient in immunoglobulins. All tested Abs were found to lose their polyspecificity in the serum environment. When in serum, the Abs no longer reacted with conventional screening Ags, including hapten‐BSA conjugates, ssDNA, thyroglobulin and myosin, but fully retained their reactivity with specific peptide Ags selected from a phage display library. The acquisition by IgM of narrow binding specificity was also observed when muscle tissue sections were used as a source of endogenous Ags. The loss of polyspecificity by different Abs was dependent on the presence of different serum constituents. The results from this study suggest that the seemingly inherent polyspecificity of natural IgM Abs is largely an in vitro phenomenon related to the lack of normal blood components in the medium.
MRL/lpr mice with a knock‐in BCR show much less efficient receptor‐editing to membrane‐bound antigen
Authors: Carey John , Lamoureux Jennifer , Feeney Ann
Published: June 2021

Receptor editing is the main mechanism by which self‐reactive BCR are removed in immature B cells. Using 3‐83 BCR transgenic mice on the MRL/lpr and B10 background, we have previously shown that receptor editing is less efficient in MRL/lpr mice using indirect measures of receptor editing such as RAG upregulation. Now we have bred the 3‐83 gene‐targeted heavy and light chain genes onto the MRL/lpr background, on both the antigenic H‐2k and non‐antigenic H‐2d background. These mice can therefore undergo normal receptor editing since the targeted light chain is in the kappa locus. Unlike the B10 3‐83 knock‐in mice, where the B cell number is similar whether self antigen (H‐2k) is present or not, we find that the spleens of MRL/lpr knock‐in mice have greatly reduced B cell numbers if self‐antigen is present. Furthermore, the level of CD19 is greatly reduced in the presence of antigen. Finally, B10 mice increase their marginal zone compartment in the presence of antigen, while MRL/lpr mice lose the MZ compartment. Thus, in the MRL/lpr mice, clonal deletion appears to be the main mechanism of eliminating B cells reactive to membrane bound antigen, while in the B10 non‐autoimmune mice, receptor editing efficiently makes a non‐self reactive normal B cell repertoire.
HuFlt3‐L treatment does not protect RIP‐GP and RIP‐NP C57Bl/6 mice against LCMV‐induced diabetes.
Authors: Van Belle Tom Lander , Ling Eleanor , von Herrath Matthias
Published: June 2021

Human Fms‐like Tyrosine Kinase3‐L (huFlt3‐L) treatment of prediabetic NOD mice has been shown to increase the intrinsically low numbers of myeloid dendritic cells in these mice, while decreasing insulitis and delaying the progression of diabetes. In this study, we investigated the influence of huFlt3‐L treatment on insulitis and diabetes onset in the LCMV‐induced RIP‐GP (fast onset) or RIP‐NP (slow onset) C57Bl/6 mouse models of T1D. We showed that these mice have no intrinsic abnormality in DC subset development during LCMV infection. As expected, huFlt3‐L treatment dramatically increased the total DC fraction and numbers in both spleen and pancreatic draining LN, with a preferential outgrowth of myeloid DCs (CD11c+B220−CD11b+) over plasmacytoid DCs (CD11c+B220+CD11b−). Moreover, NK cells and NK‐DC cells were also increased, but CD4 and CD8 T cell fractions were reduced. Strikingly, repeated huFlt3‐L administrations, initiated in the prediabetic phase, did not protect against diabetes onset in this model. It is possible that this is correlated with the observed reduced fraction of CD4+CD25+ T cells in the huFlt3‐L‐treated mice. Translation of these data suggests that huFlt3‐L might only be a useful therapeutic agent for T1D patiens with a demonstrated DC (subset) defect.
CD11c+CD11b+ dendritic cells play an important role in intravenous tolerance and suppress ongoing experimental autoimmune encephalomyelitis
Authors: li hongmei , zhang Guang xian , Rostami abdolmohamad
Published: June 2021

The central role of T cells in the induction of tolerance against experimental autoimmune encephalomyelitis (EAE) has been well documented. However, the role of dendritic cells (DCs), the most potent antigen presenting cells, in this process is not clear. In the present study we address this important question, with particular focus on the differential roles of DC subsets, CD11c+CD11b+ and CD11c+CD8+ DCs, in the induction of tolerance. We found that the proportion of CD11c+CD11b+ DCs was increased in the central nervous system (CNS) and spleen of mice injected intravenously (i.v.) with autoantigen peptide MOG35‐55, which developed significantly less severe EAE than PBS‐injected control mice. These DCs exhibited immunoregulatory characteristics, such as increased production of IL‐10, inhibition of MOG‐induced T cell proliferative responses, and induction of CD4+CD25+Foxp3+ regulatory T cells. More importantly, these DCs significantly suppressed ongoing EAE upon adoptive transfer. Our observations demonstrate that CD11c+CD11b+ DCs, which are abundant in the CNS during induction of tolerance, are crucial for i.v. tolerance induction in EAE and could be exploited for immunotherapy of autoimmune diseases.
The effects of aging on T and B cell tolerance to AChR in myasthenia gravis
Authors: Stacy Sue , Williams Earlanda , Krolick Keith A. , Kraig Ellen
Published: June 2021

Autoimmune myasthenia gravis (MG) is a T cell dependent, B cell mediated disease in which symptoms of muscle weakness often show a late age of onset. Thus, it has been suggested that tolerance to muscle acetylcholine receptor (AChR) may weaken with age. To address this, we developed a murine transgenic model in which the Torpedo californica AChRα chain (TAChRα) is expressed as a neoself antigen. This results in T cell tolerance, as evidenced by diminished proliferation to alpha chain epitopes. Surprisingly, this T cell tolerance to TAChR was maintained in old transgenic animals and could not be broken by multiple immunizations or by IL12. Moreover, “old” transgenic lymph node cells (LNCs) produced reduced levels of IL2, INFγ, and TNFα, relative to LNCs from nontransgenic mice; the same pattern was observed in young animals. To assess the effects of aging on autoantibody responses, the sera from old TAChR‐immunized mice were tested for reactivity to recombinant Torpedo or mouse AChRα chains. An age‐associated loss of reactivity was seen and there was no difference in the response to self (mouse) epitopes. Thus, mice were then immunized with recombinant TAChRα chain. This eliminated bystander help and revealed that the TAChR transgenic animals did exhibit B cell tolerance to TAChRα; reduced titers of induced TAChRα reactive antibodies were seen. In old mice, the immune responses were very low, but there was no detectable loss of tolerance. Thus, our data suggest that aging dysfunction alone is not sufficient to break B cell and T cell tolerance to AChR. Support: Myasthenia Gravis Foundation, Nathan Shock Aging Center, NIH R03 AG 14557 and San Antonio Area Foundation.
Selenium affects allergic asthma by regulating CD4+ T cell activation and dendritic cell maturation
Authors: Hoffmann Peter R , Hoffmann FuKun , Jourdan‐LeSaux Claude , Bollt Oana , Tam Elizabeth , Berry Marla J
Published: June 2021

To assess the affect of dietary selenium (Se) on the development of asthma, we fed diets containing low (0.08 ppm), medium (0.25 ppm), or high (2.7 ppm) Se to mice and used an established OVA challenge protocol to induce allergic asthma. Results demonstrated that mice fed medium levels of Se had the highest levels of asthma. In contrast, responses to OVA challenge were less robust in mice fed low or high levels of Se. To evaluate potential mechanisms by which Se affects allergic asthma, we measured the effect of Se levels on activation of CD4+ T cells and maturation of dendritic cells (DCs). Consistent with the asthma results, medium Se led to highest levels of proliferation and increased expression of Th2 marker, OX40. Medium Se also led to highest increases in the DC2 marker, OX40L. Interestingly, increasing Se levels correlated with increased percentage of CD25hiFoxP3+ cells and decreasing RANKLhi expression, both consistent with a Treg phenotype. Overall, our results suggest that Th2‐type responses, like those that drive allergic asthma, are influenced by dietary Se levels through a mechanism that involves interactions between DCs and CD4+ T cells mediated by OX40‐OX40L. Moreover, increased Se intake may lead to increased numbers of Treg cells that arise during the initiation of immune responses, and this may be a mechanism by which Se intake regulates a wide variety of immune responses.
Atropine‐induced potentiation of airway hyperreactivity in antigen challenged guinea pigs is mediated by nerve growth factor
Authors: Verbout Norah G , Jacoby David B , Fryer Allison D
Published: June 2021

Eosinophils accumulate around airway nerves and cause airway hyperreactivity in antigen challenged guinea pigs. Anticholinergic drugs such as atropine reverse airway hyperreactivity in challenged animals. Similarly, anticholinergics reverse asthma exacerbations in man; however, they are less effective in managing chronic asthma. Conversely, atropine administration to sensitized guinea pigs 1h before challenge significantly potentiates airway hyperreactivity measured 24h later and is mediated by eosinophils. Since eosinophils produce nerve growth factor (NGF), we tested whether NGF causes atropine‐potentiated hyperreactivity. Sensitized guinea pigs were given antibody to NGF (AbNGF) 1h before challenge. Electrical stimulation of both vagus nerves caused bronchoconstriction that was potentiated in challenged animals. Atropine pretreatment further potentiated vagally induced bronchoconstriction in challenged animals, an effect blocked by AbNGF. Atropine‐induced increase in eosinophil protein deposition (assessed by immunohistochemistry) in challenged animals was blocked by AbNGF. Thus, NGF mediates atropine‐induced potentiation of airway hyperreactivity, possibly due to increased eosinophil activation. These data suggest that anticholinergics enhance eosinophil interactions with airway nerves via neurotrophins.

NIH HL55543 HL54659 ES014601 HL071795 AHA 0515486Z
Protection from vaccinia virus‐induced severe skin lesions by natural killer cells in a mouse model of eczema vaccinatum
Authors: Kawakami Yuko , Hasegawa Shunji , Yumoto Kenji , Yao Libo , Tomimori Yoshiaki , Tagaya Yutaka , Crotty Shane , Kawakami Toshiaki
Published: June 2021

Threats of bioterrorism have recently renewed efforts to better understand the disease mechanism of smallpox and to develop a safer vaccine against this dreadful disease. Individuals with atopic dermatitis (AD) are excluded from smallpox vaccination due to their propensity to develop eczema vaccinatum, a disseminated vaccinia virus infection. To study the underlying mechanism of the vulnerability of atopic patients to vaccinia virus infection, we developed a mouse model of eczema vaccinatum by infecting eczematous skin with virus. Virus infection induced severe erosive skin lesions in mice with preexisting dermatitis, but not in mice with healthy skin. The former mice exhibited lower natural killer (NK) cell activity, but similar cytotoxic T lymphocyte activity and humoral immune responses, as compared to the latter mice. The role for NK cells in suppressing vaccinia virus‐induced development of erosive and satellite skin lesions was demonstrated by depletion and adoptive transfer of NK cells. Consistent with low NK cell activities in AD patients, these results can at least partly explain the increased susceptibility of AD patients to eczema vaccinatum.

Funded by the NIH through the ADVN consortium.
Circulating regulatory T‐cells: Acute exercise response in elite swimmers
Authors: Wilson Lori Doss , Schwindt Christina D , Zaldivar Frank P , Cooper Dan M
Published: June 2021

Several authors have found higher rates of allergy and asthma (AA) among elite endurance athletes. The purpose of this study was to characterize the pattern of Regulatory T‐cells (Tregs) and FoxP3+ Tregs at baseline (Pre) and at the end of high‐intensity swim exercise (EE). On the weekend after their last high school swim meet, 22 elite swimmers (12 males, mean age=15.4 years, AA group=12) participated in a swim study. Spirometry was measure before and 10–15 minutes post EE and blood collected Pre and EE. Neither Pre nor EE blood lactate, FEV1, heart rate, or lymphocyte subsets (% of cells or absolute counts, #s) differed between the groups (control vs AA). Controls had lower CD4+CD25+FoxP3 dim cells (% of lymphs) than the AA group (p<0.05), though absolute cell counts did not differ. As shown on Figure 1, the AA group had higher percentages and absolute FoxP3+ cell counts (n.s.) at both Pre and EE. Absolute cell counts increased in both groups during exercise however, the percentages of FoxP3+ cells decreased from Pre to EE. An ancillary finding of this study was that the baseline levels of Tregs and FoxP3+ cells in the elite swimmers appeared to be substantially lower than values previously reported for non‐endurance athletes. This study was funded by USA Swimming and NIH P01 HD048721.
Endotoxin Tolerance Dysregulates MyD88‐Dependent and TRIF‐Dependent Signaling Pathways and Increases Expression of Negative Regulators of TLR Signaling
Authors: Medvedev Andrei E. , Chen Haiyan , Piao Wenji
Published: June 2021

Endotoxin tolerance represses expression of proinflammatory cytokines and chemokines while not affecting anti‐inflammatory cytokines and anti‐microbial effectors (reprogramming). Septic shock patients often develop increased susceptibility to infections and decreased responsiveness to LPS, which closely resembles the endotoxin‐tolerant phenotype. Little is known regarding molecular mechanisms of induction and maintenance of endotoxin tolerance. In this study, we examined LPS‐mediated activation of MyD88‐dependent and TRIF‐dependent pathways, as well as expression levels of negative regulators of TLR signaling in control and endotoxin‐tolerant human monocytes. Tolerization resulted in decreased LPS‐inducible dimerization, phosphorylation and nuclear translocation of interferon regulatory factor (IRF)‐3. Consistent with the importance of IRF‐3 for activation of MyD88‐independent, TRIF‐dependent genes, real‐time PCR analyses of endotoxin‐tolerant cells revealed suppressed expression of RANTES and IFN‐β mRNA in response to LPS. Reduced LPS‐inducible IRAK‐1 and p38 phosphorylation, IκB‐α degradation, and nuclear translocation of IRF‐5 noted in LPS‐tolerant cells were accompanied by inhibited TNF‐α and IL‐8 mRNA expression. Real‐time PCR and immunoblotting analyses of negative regulators of TLR signaling documented increased expression levels of Tollip mRNA and protein, as well as accumulation of SOCS‐1, SHIP‐1, IRAK‐M, and ST‐2 mRNA in endotoxin‐tolerant monocytes. Taken together, these data demonstrate TLR4‐triggered MyD88‐dependent, TRAM/TRIF‐dependent intermediates, transcription factors, cytokines, and regulators of TLR signaling that are subject to modulation by endotoxin tolerance.

This work was supported by NIH grant AI‐059524 (to A.E.M.).
Extracellular ATP induces IL‐1beta secretion and formation of filopodia in myeloid lineage cells through distinct P2X7‐dependent pathways
Authors: Thomas Louis Michael , Chu Jessica , Watkins Simon C. , Stolz Donna , Salter Russell D.
Published: June 2021

Extracellular ATP (ATPe) is an endogenous danger signal which acts as a ligand for purinergic receptors on immune cells, resulting in complex changes in cytokine production, migration, function, and morphology. We have identified cytoskeletal changes in macrophages and dendritic cells (DC) induced by ATPe acting on P2X7, an ion channel purinergic receptor, that result in formation of unbranched non‐substrate attached filopodia (AIF) that frequently fragment into microvesicles. We investigated whether these structures represent a source of inflammatory cytokines including IL‐1beta, whose release is known to be induced by ATPe via P2X7 activation. Pharmacologic and genetic manipulations to probe both IL‐1beta secretion and AIF formation demonstrated that the pathways controlling these two processes are distinct. AIF formation requires actin polymerization and PLD activity while being insensitive to inhibition of pannexin and caspase‐1 function. In contrast, IL‐1beta secretion is not blocked by inhibitors of PLD, but does require caspase‐1 and pannexin activity. These results show that extracellular ATP induces a complex response in myeloid cells involving multiple signaling pathways.
HIV‐1 ssRNA triggers a vitamin D‐dependent anti‐viral pathway in human monocytes
Authors: Krutzik Stephan R , Hong Patrick , Robles Antonio , Corcoran Kellie , Modlin Robert L , Lee Benhur
Published: June 2021

Toll‐like receptors mediate the activation of critical host defense mechanisms such as cytokine/chemokine secretion and a vitamin D‐dependent anti‐microbial pathway in response to pathogens. Here we investigated whether TLR8, which mediates cellular activation by HIV‐1 ssRNA, triggers these two key defense mechanisms in human monocytes. We found that HIV‐1 ssRNA and a synthetic TLR8 ligand stimulates monocytes to secrete IL‐12 as well as the CCR5 ligands RANTES, MIP1α and MIP1β. Furthermore, HIV‐1 ssRNA triggered gene expression of two critical components of the vitamin D‐dependent anti‐microbial pathway, CYP27b1 (1‐α hydroxylase) and the vitamin D receptor, which together enables monocytes to induce gene expression of the anti‐microbial peptide cathelicidin. Pretreatment of HIV‐1 with cathelicidin peptide directly inhibited HIV‐1 infectivity. Finally, the active vitamin D hormone 1,25‐dihydroxyvitamin D3 triggered cathelicidin gene expression in HIV‐1 infected monocytes and induced an anti‐viral activity against HIV‐1. The 1,25D3‐induced anti‐viral activity was ablated by a cathelicidin specific siRNA. In conclusion, activation of human monocytes with HIV‐1 ssRNA triggers 1) release of cytokines/chemokines with known roles in HIV host defense and 2) a vitamin D‐dependent anti‐viral pathway that is mediated by cathelicidin.
Critical role of IgG receptors (FcγRs) in LPS‐induced severe lung inflammation
Authors: Krupa Agnieszka , Fudala Rafal , Stankowska Dorota , Kurdowska Anna
Published: June 2021

Previously we showed that anti‐IL‐8:IL‐8 complexes (IL‐8 associated with anti‐IL‐8 autoantibodies) present in lungs of patients with ALI/ARDS correlate with both the development and outcome of ALI/ARDS. We also demonstrated that anti‐KC autoantibody:KC immune complexes (KC ‐ functional IL‐8) are present in the alveolar spaces of mice treated with LPS. Activity of these complexes is mediated by IgG receptors (FcγRs) because alveolar inflammatory response was substantially diminished in γ‐chain deficient (KO) mice that lack these receptors. In the current study we established that the expression of TLR4 and FcγRs is increased in lungs of mice treated with LPS, and that these receptors partially co‐localize. Further, Yes/Src was activated in neutrophils obtained from LPS treated mice at 6 and 14 h but not 2h, and was not detected in KO mice. Thus, activation of Yes at 6 and 14 h is a consequence of engagement of FcγRs by anti‐KC:KC complexes which are formed in lungs at around 6 h after LPS administration, and is unique for the FcγR signaling pathway. In contrast, we detected phosphorylated Syk already at 2 h since Syk is also activated through TLR4. Lack of activated Syk in KO mice at 6 and 14h indicates that activation of Syk is induced via FcγRs, by the anti‐KC:KC complexes that appear in the lungs at 6 h. Our results suggest a possible crosstalk between TLR4 and FcγRs in lung inflammation.

Supported by the NIH grant HL073245.
Toll‐like Receptor 9 constitutively traffics from the ER to the lysosome prior to stimulation with CpG DNA
Authors: Chockalingam Annapoorani , Lopez Jody L , Brooks James C , Leifer Cynthia A
Published: June 2021

The innate recognition of unmethylated CpG dinucleotide motifs present in microbial DNA is mediated through Toll‐like receptor 9 (TLR9). This immunostimulatory effect has been harnessed for therapeutic purposes in vaccines as adjuvant and in cancer therapy. However, recognition of self DNA complexes by TLR9 leads to autoimmune pathogenesis. Control of TLR9 localization contributes to discrimination between self and microbial DNA. The objective of our research is to elucidate the molecular mechanism that regulates TLR9 localization and trafficking. To determine if TLR9 traffics constitutively through the secretory pathway, we developed two in‐vitro assays. A TLR9 chimeric construct, with a furin cleavage site, demonstrates that TLR9 constitutively traffics to the Golgi. Lectin blotting of TLR9 glycan modifications indicates that TLR9 traffics through the Golgi to reach the trans‐Golgi network. Cell fractionation experiments further demonstrate that TLR9 constitutively traffics to lysosomes. Constitutively trafficked TLR9 may initiate signaling in response to CpG DNA since the Golgi disrupting agent Brefeldin A inhibits late, but not early TLR9 signaling events. Together, our data support a model where TLR9 constitutively traffics through Golgi to reach lysosome where signaling is initiated in response to CpG DNA. Supported by NIH grant K22CA113705 awarded to C.A. Leifer.
Regulation of Kv1.3 expression by C5b‐9 terminal complex in oligodendrocytes and their role cell cycle activation
Authors: Cudrici Cornelia Diana , Soloviova Kateryna , Rozycka Monika , Tegla Cosmin Adrian , Judge Susan , Rus Horea
Published: June 2021

Proliferative oligodendrocytes progenitors cells (OPC) express various potassium (K+) channels including Kv1.3 required for the G1/S transition. In contrast, mature oligodendrocytes (OLG) exhibit few Kv1.3 channels. We examined Kv1.3 expression in OLG and the effect of C5b‐9 on Kv1.3 expression. Kv1.3 was found to be expressed in rat OPC, with differentiated OLG exhibiting little Kv1.3. Exposure to purified C5b‐9 significantly increased Kv1.3 expression while C5b6 had no effect. To test functionality, we blocked Kv1.3 in C5b‐9 stimulated OLG using the recombinant scorpion toxin rOsK‐1 (Orthochirus scrobiculosus) and found that this K+ channel blocker abolished Kv1.3 expression. Kv1.3 expression was also inhibited by Pertussis toxin indicating the requirement of Gi‐protein. Interestingly, Akt phosphorylation and activation by C5b‐9 was also inhibited by rOsK‐1 strongly suggesting a role for Kv1.3 in controlling C5b‐9 downstream signaling pathways. Since Akt play a major role in C5b‐9 induced cell cycle activation we investigated the effect of inhibiting Kv1.3 on DNA synthesis. Pretreatment with rOsK‐1 significantly inhibited 3[H]‐Thymidine incorporation induced by C5b‐9 in OLG, indicating that Kv1.3 plays an important role in C5b‐9 induced DNA synthesis. In conclusion, our data suggest that Kv1.3 plays an important role in C5b‐9 OLG cell cycle activation and survival in an inflammatory milieu.
Macrophage/neutrophil specific Arginase‐1 is a critical survival factor and regulator of liver fibrosis during Schistosoma mansoni infection
Authors: Pesce John T , Cheever Allen W. , Ramalingam Thirumalai R. , Wilson Mark S. , Mentink‐Kane Margaret M. , El Kasmi Karim C. , Smith Amber M. , Murray Peter J. , Wynn Thomas A.
Published: June 2021

Fibrosis is a contributing factor to morbidity and mortality associated with several diseases. Infection with S. mansoni results in parasite egg deposition within the liver inducing high levels of IL‐4 and IL‐13, causing granulomatous lesions to form around the egg. In addition, these cytokines are responsible for the differentiation of resting macrophages into alternatively activated macrophages (AAMΦ) that express Arginase‐1 (Arg‐1), which are a major population within the granuloma. In these studies, mice with a macrophage/neutrophil‐specific conditional knockout of the Arg‐1 gene were infected with S. mansoni for up to 22 weeks. The absence of Arg‐1 during infection with S. mansoni resulted in 40% mortality whereas 100% of littermate controls survived to the chronic stage of infection. Death was not due to excess nitric oxide production as oral treatment with aminoguanidine failed to reduce the mortality. Histological examination of liver sections showed a significant increase in granulomatous inflammation in the LysMCreArg‐1flox/flox KO mice, which was associated with increased collagen deposition and mast cell infiltration. Observed variations in granuloma volume and fibrosis may be attributed to small yet consistent increases in IL‐13 production over the course of the infection. These data suggest that Arg‐1 is a critical regulator of survival and infection‐induced pathology during S. mansoni infection.
IL‐23 aids in defense against Francisella infection
Authors: Butchar Jonathan P. , Rajaram Murugesan V.S. , Parsa Kishore V.L. , Clay Corey D. , Gunn John S. , Schlesinger Larry S. , Tridandapani Susheela
Published: June 2021

Francisella tularensis is the causative agent of tularemia and is classified as a Category A bioterrorism threat. It primarily invades monocytes and macrophages, where it escapes the phagosome and replicates in the cytosol. We performed a microarray screen to study human monocyte responses to F. tularensis novicida, a related subspecies lethal to mice. We identified IL‐23, a recently discovered cytokine related to IL‐12, and confirmed its upregulation using Western blotting and ELISAs. Functional studies showed that this infection‐induced IL‐23 could elicit IFNg production in natural killer cells. Hence, IL‐23 is important during infection, since IFNg is needed for IL‐12 production and blockage of both IL‐23 and IL‐12 leads to higher infectivity.

Monocyte infection with virulent Francisella (SCHU S4) also led to IL‐23 production, but at much lower levels than from F. novicida. Further studies showed that IL‐23 production requires PI3 Kinase and NFkB activation, which are negatively regulated by the inositol phosphatase SHIP. Interestingly, SHIP was downregulated by F. novicida but not by SCHU S4 infection. This may account for the reduced IL‐23 response to the more virulent strain.
Inhibition of phago‐lysosome fusion by chlamydial elementary bodies is necessary for the early induction of type I IFNs in mouse macrophages
Authors: Nagarajan Uma M , Wyrick Priscilla B , Rank Roger G
Published: June 2021

Two strains of Chlamydia, the mouse Chlamydia muridarum (MoPn) and the guinea pig Chlamydia caviae (GPIC) have equivalent growth rates in epithelial cells while only MoPn grows effectively in macrophages. To determine the early gene expression differences in mouse macrophages to these 2 organisms, micro array analysis was performed from RNA collected 3h post infection, followed by confirmation by real‐time quantitative RT‐PCR. Uninfected cells and cells treated with UV‐inactivated MoPn or GPIC were used as controls. Expression of pro‐inflammatory cytokines TNF‐α, IL‐6, IL1β, and inhibin β‐A expression were similar in GPIC or MoPn infected macrophages. However, only MoPn infection induced the expression of type I IFN and IFN response genes such as Egr‐1, IP‐10, and Gem GTPases. These responses were not observed in GPIC‐infected macrophages, or in UV‐inactivated MoPn and GPIC. To address the role of early bacterial and host interaction in the differences in gene expression, the infected macrophages were studied by electron microscopy (EM). EM analyses show the presence of intact chlamydial elementary bodies (EB) and reticulate bodies (RB) in endosomes of MoPn‐infected macrophages after 2h. In contrast, the GPIC EB's ended up in phagocytic vacuoles suggesting these EBs were killed. These data suggest that escape of MoPn chlamydial EB from lysosomal fusion is critical for the initiation of type I IFNs and IFN response genes. Further, these data suggest that induction of type I IFNs could be mediated by innate receptors interacting with the early endosomes containing chlamydiae.
IL‐7 enhances human antigen specific memory T‐cell responses: Importance for improved diagnosis of TB infection
Authors: Feske Marsha Lynn , Medina Miguel , Lew Justin , Nudelman R , Graviss Edward A , Lewis Dorothy E
Published: June 2021

T‐cell interferon‐gamma (IFN‐γ) release assays (TIGRAs) use ELISA and ELISPOT assay techniques and are being used in place of the century‐old skin test (TST) to diagnose latent and clinical TB infection in some adult populations. TIGRA's have a greatly increased diagnostic specificity, compared to TST. This test however, is not predictive in HIV infected or immunocompromised adults, or in children. In attempt to improve IFN‐γ detection, Interleukin 7 (IL‐7), a survival cytokine known to enhance viability and stabilize IFN‐γ message, was used in cells from three different groups of adult subjects: Tetanus immunized control group, TB positive patients, and HIV positive patients. IFN‐γ production was measured with and without IL‐7 addition using both ELISA and ELISPOT techniques. IL‐7 addition to antigen stimulated PBMC's resulted in up to a 14‐fold increase (μ=4), as measured by ELISA. Increased IL‐7 responses in tetanus controls, HIV positive and TB infected patients were statistically significant: p<0.001, p =0.08 and 0.05 respectively. ELISPOT results allowed a count of effector cell number, and confirmed ELISA findings: p<0.01, p=0.03 and p=0.02. A strong correlation was seen between ELISA and ELISPOT in each individual (R2=0.82–0.99). Increased detection of IFN‐ γ may further increase sensitivity and specificity for testing of TB and therefore increase the use of TIGRA's in populations with high TB risk.
Nutrient and food group intake from school meals and bag lunches consumed by school‐aged children
Authors: Hur Inyoung , Burgess‐Champoux Teri , Reicks Marla
Published: June 2021

School meals can be an important source of energy and specific nutrients as well as fruits and vegetables. Studies comparing intake from lunch provided at school and lunches carried from home are limited. The purpose of this study was to examine the contribution that school meals made to nutrient, fruit and vegetable intake and meal energy density of children. Lunchroom meal observation procedures were used to collect food intake data from 129 children (ages 8–12 yr) in a suburban Midwestern school. Dietary analyses were completed using Nutrition Data System for Research software and t‐test and general linear models analysis were used to compare nutrient, and fruit and vegetable intake. Children eating school lunch (n=85) had a higher intake of protein, vitamins A, D, and K, and calcium, and a lower intake of energy, fat, carbohydrate, vitamin E and added sugars compared to those who brought a bag lunch from home (n=44). The ratios of mean protein and calcium consumption per minimum values required by USDA regulations for school lunch tended to be higher for children eating school lunch than those eating bag lunches. Children eating bag lunches had a lower fruit and vegetable intake and higher energy density than children eating school lunch. Overall, children who ate school lunch had a more favorable macro and micronutrient and fruit and vegetable intake compared to those who brought a bag lunch from home.
Nutrition status improvement of low income family toddlers with nutrition education and supplementary food
Authors: Jeon Joo‐Youn , Cha Youn‐Soo
Published: June 2021

This study was performed to evaluate the effectiveness of nutrition education and providing of supplementary food for low‐income family toddlers. The subjects in this study consisted of 116 toddlers and nutrition education and supplementary food were provided for 6 months. The results are as follows. The pre and post height and weight means were 93.88cm, 14.14kg and 96.46cm, 15.12kg and showed significant difference after providing nutrition education and supplementary food for 6 months (p<0.001). Pre and after Hemoglobin value, 11.13g/dl to 11.73g/dl were significantly increased after providing nutrition education and supplementary food (p<0.001). The nutrition knowledge scores and dietary habits were increased and show significant difference after providing nutrition education and supplementary food for 6 months. The intake of protein and fat was significantly increased after providing nutrition education and supplementary food (p<0.001). Vitamin C intake was decreased but the difference was not significant. It was concluded that providing nutrition education and supplementary food for 6 months can improve nutritional status of low income toddlers as well as nutrition knowledge and dietary habits their mothers.