bluuebunny/crossref_metadata_2025 huggingface dataset:
Antagonism of MAVS Signaling by Rotavirus Infection
Authors: Ding Siyuan , Greenberg Harry B.
Published: January 2023

The single most important cause of severe gastroenteritis and diarrhea in young children, rotaviruses (RVs) are responsible for an annual 450,000 deaths worldwide. Despite the availability of several vaccines, RV global mortality is still high and significantly less effective in the poorest countries where the burden of disease is greatest. Therefore, there remains an urgent need for a deeper understanding of the basic mechanisms of RV pathogenesis and host-pathogen interaction to develop improved vaccines and novel antiviral therapeutics. The host protein MAVS plays a pivotal role in restricting the replication of many RNA viruses by relaying antiviral signaling from the cytoplasmic RNA sensors RIG-I and MDA5 to downstream interferon (IFN) induction. Multiple virus-encoded factors, including HCV NS3/4A, antagonize the antiviral activity of MAVS by proteolytic cleavage. We recently observed that RV infection specifically mediates the degradation of full-length MAVS but not the truncated variant mini-MAVS. This process is independent of well-characterized IFN antagonist RV NSP1. Given the importance of IFN in suppressing the replication of certain RV strains in vivo, we hypothesize that RVs utilize one or more viral proteins to dampen IFN responses by targeting MAVS signaling. We will mechanistically determine the viral and host factors responsible for MAVS degradation and examine whether it takes place in intestinal epithelial cells in vivo. The current study will broaden our knowledge of RV pathogenesis and provide a molecular basis for the rational design of a new-generation of attenuated RV vaccines.
Chronic chikungunya virus infection disrupts the architecture and function of draining lymphoid tissue
Authors: McCarthy Mary K , Hawman David W , Elmore Susan A , Morrison Thomas E
Published: January 2023

Chikungunya virus (CHIKV) causes large outbreaks of rheumatologic disease, resulting in chronic disease symptoms in up to two-thirds of infected individuals. Studies in humans and animal models suggest that CHIKV infection persists in musculoskeletal tissues, although the mechanisms are unclear. The pathogenic clinical isolate AF15561 persists in joint tissue of WT mice, while the attenuated strain 181/25 is cleared. In contrast, 181/25 persists in joint tissue of Rag1−/− mice at levels similar to AF15561, suggesting that adaptive immune responses are critical for clearance of 181/25 but are impaired during AF15561 infection. We hypothesized that early events in lymphoid tissue during persistent or acute CHIKV infection are distinct and these differences contribute to CHIKV persistence or clearance in joint tissue. To test this hypothesis, we performed functional and histological analyses of the draining lymph node (dLN) during acute and persistent CHIKV infection. In mice infected with 181/25, we detected activation of CD21+CD23+ conventional follicular B cells and increased numbers of Tfh cells. In contrast, activated B cells in the dLN of AF15561-infected mice were CD21lo/−CD23−and Tfh cell numbers were reduced. Acute 181/25 infection also resulted in increased cortex area, plasma cells, and germinal center (GC) formation in the dLN, while the dLN of AF15561-infected mice was depleted of lymphocytes and became fibrotic. Following NP-OVA immunization, development of NP-specific GC B cells was decreased during AF15561, but not 181/25 infection, suggesting that persistent CHIKV infection impairs dLN function. These data provide new insight into the immunologic mechanisms that facilitate CHIKV persistence in joint tissue.
An imbalance between MMPs and TIMPs plays a critical role in the pathogenesis of HIV-associated neurocognitive disorders
Authors: Xing Yanyan , Shepherd Nicole , Lan Jie , Yang Kai , Amet Tohti , Dong Jun , Yu Qigui
Published: January 2023

HIV-associated neurocognitive disorders (HAND) affects approximately 50% of HIV-infected individuals, and disruption of the blood brain barrier (BBB) has been observed in individuals with HAND. One of the underlying mechanisms of BBB breakdown is an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), which has not been fully explored in HAND. In our present study, we used ELISA and zymography assays to examine the protein levels and enzyme activities of MMPs and/or TIMPs in plasma from a total of 59 HIV-positive individuals and 16 uninfected controls. We found that the concentration and enzyme activity of MMP-2 and MMP-9, two of the most important members of the MMP family, were increased in the plasma of HIV patients compared to HIV-negative controls. Plasma levels of TIMP-1 and TIMP-4 were not affected, while TIMP-3 was significantly increased in HIV patients. Strikingly, TIMP-2 was significantly down-regulated in HIV-1 patients. We also showed that neural cells, especially astrocytes, were able to release both MMPs and TIMPs, and such secretion was regulated by inflammatory factors like IL-1β or growth factors like TGF-β1. Furthermore, we found that TIMP-2 played a dual role on proMMP-2 activation via the ternary complex of MT1-MMP/TIMP-2/proMMP-2 on the surface of astrocytes, as low concentrations of TIMP-2 activated proMMP-2 and high concentrations inhibited proMMP-2 activity. In summary, our results demonstrate that decreased TIMP-2 found in HIV patients enhances MMP-2 activity, which is associated with BBB breakdown in HIV patients. Exogenous administration of TIMP-2 to maintain MMPs/TIMP-2 homeostasis may represent a potential therapy for HAND.
Liver-associated macrophage infiltration and altered cellular signaling in SIV-infected macaques.
Authors: Fisher Bridget , Arang Nadia , Klatt Nichole R , Kaushansky Alexis , Sodora Donald
Published: January 2023

Liver disease remains an important contributor to morbidity and mortality in HIV-infected patients. In addition to the effects of HIV replication, bacterial translocation from the gastrointestinal tract potentially contributes to liver dysfunction. Indeed, bacteria leaving the gut filter through the liver via portal vein circulation. Providing patients with combination anti-retroviral therapy (cART) does provide some reduction to the generalized immune inflammation, but also can induce liver toxicity. The SIV macaque model recapitulates HIV immune activation and bacterial translocation and was utilized here to evaluate livers of uninfected (n=3), SIV-infected (n=5), and SIV-infected/cART-treated rhesus macaques (n=6). Using immunofluorescence microscopy, CD68+ macrophages (associated with inflammation) were demonstrated to increase in livers of SIV+ macaques, but normalize in cART-treated SIV+ macaques. In addition, reverse-phase protein arrays (RPPA) identified a number of signaling proteins that were activated (phosphorylated) in SIV+ macaques, including proteins involved in the Wnt/beta-catenin pathway (e.g. Dvl-3, pGSK3αβ) as well as inflammatory STAT (e.g. pSTAT2) and NFkB signaling proteins. Assessment of SIV+/cART-treated macaques determined that the elevation in these same signaling pathways could be observed even after 7–8 months of cART. Collectively, this suggests that although cART treatment can reverse macrophage infiltration in the liver during SIV infection, it does not restore normal hepatocyte cellular signaling networks. We conclude that SIV infection results in liver immune activation and that residual liver inflammation persists following cART administration.
Evasion of the African green monkey protein kinase R pathway by mutation of vaccinia virus RNA polymerase
Authors: Brennan Greg , Kitzman Jacob O. , Shendure Jay , Geballe Adam P.
Published: January 2023

One of the earliest barriers to cross-species transmission is mediated by host restriction factors, a group of antiviral proteins that have evolved to detect and then inhibit viral replication. To productively infect a new species, viruses must rapidly adapt to overcome host-specific orthologs of these restriction factors. To model this process we created a recombinant vaccinia virus (VACV), replacing its antagonists of the host restriction factor PKR with the rhesus cytomegalovirus PKR antagonist RhTRS1 (VACVΔEΔK+RhTRS1). Relative to wildtype virus, VACVΔEΔK+RhTRS1 replication was reduced 100-fold in primary African green monkey (AGM) fibroblasts. This replication defect was mediated entirely by PKR. After several rounds of serial passage, we found that genetic amplification of rhtrs1 rescued VACVΔEΔK+RhTRS1 replication in AGM cells. Further serial passage identified mutations in two VACV genes that were individually sufficient to rescue VACVΔEΔK+RhTRS1 replication in AGM cells. Fixation of these mutations coincided with collapse of the rhtrs1 amplification. One of these mutations occurred in the catalytic subunit of the viral RNA polymerase, A24R, and provided a replication benefit independent of rhtrs1. Different mutations in A24R have been shown to prevent activation of RNase L by reducing dsRNA production during viral infection. However, the A24R mutation that evolved during this serial passage does not prevent RNase L activation in AGM cells. Furthermore, this mutation does not decrease the total concentration of dsRNA produced during infection. This work identifies new PKR evasion genes, and suggests that the viral RNA polymerase is an important modulator of multiple host innate immune responses during infection.
HTLV-1 infection and neuropathogenesis in the context of Rag1−/−γc−/− (RAG1) and BLT mice
Authors: Jain Pooja , Ginwala Rashida , Charlins Paige , Akkina Ramesh , Loonawat Ronak , Caruso3 Breanna , Jacobson Steven , Sreedhar Sreesha , Khan Zafar
Published: January 2023

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a disabling chronic inflammatory disease of the central nervous system (CNS) with similarities to multiple sclerosis (MS). To date, the lack of a suitable small animal model has hindered our quest to understand the immuno- and neuropathogenesis of HTLV-1 in an in vivo system. Previous work from others have established that host immune response plays a critical role in the outcome of HTLV-1 infection, which could be better tested in the context of humanized (hu) mice. Thus, we employ here the Rag1 as well as Bone marrow-Liver-Thymic (BLT) mouse models for engraftment of human CD34+ hematopoietic stem cells and followed HTLV-1 infection. Flow cytometry and histological analyses revealed reconstitution of Rag1 and BLT mice with human immune cells, including macrophages, dendritic cells, T cells and B cells. Proviral load (PVL) was determined in the peripheral blood, spleen, and other organs of neonatal and adult Rag1 and BLT hu-mice by droplet digital PCR. Tax showed peak expression at 14 wpi in Rag1 mice with continued expression until 16 weeks. Within blood, PVL and viral protein Tax was detected as early as 2 wks post-infection (wpi) in Rag-1 and BLT hu-mice. Successful infection was followed by immune activation and Tax expression within lymphoid organs. Moreover, lymphocytic infiltration with concomitant Tax expression and resulting myelin disruption were observed in the spinal cord of the infected mice. These data represents the first attempt to establish HTLV-1 neuropathogenesis in the context of RAG1 and BLT mice suggesting possibility of developing a small animal model of HAM/TSP in humanized mice.
Blockade of proteasomal degradation enhances MHC-II dependent peptide epitope production through chaperone dependent autophagy in dendritic cells infected with Mycobacterium bovis BCG vaccine
Authors: Khan Arshad , Moruno Jose Felix , Tsvetkov Andrey , Jagannath Chinnaswamy
Published: January 2023

Bacillus Calmette Guerin (BCG) is the most frequently used and safe live attenuated vaccine against human tuberculosis. BCG secretes antigen-85B (Ag85B) which is an immunodominant protein generating protective CD4 T cell responses against tuberculosis. We found that over-expression of Ag85B in BCG (85B-BCG) led to aggresome formation in the cytosol of primary mouse dendritic cells. Aggresomes were cleaved through both proteasomes and lysosomes since, blockade of proteasomes using bortezomib led to an increase in the presentation of the p25-epitope of Ag85B to CD4 T cells, which secreted IL-2. We demonstrate here that during the initial stages of aggresome formation, much of the secreted Ag85B is lost through proteasomal degradation while over-expression led to the ubiquitination of Ag85B containing aggresomes. Using siRNA knockdown and biochemical inhibition for protein sorting agents, we demonstrate here that ubiquitinated Ag85B complexes recruit ‘co-chaperone Carboxyl Terminus HSP70/90 Interacting Protein’ (CHIP), which in turn binds to hsp70 triggering a cascade where hsp70 binds to bcl2-associated athanogene-3 (BAG3) followed by p62. The latter culminates in LC3 activation and formation of an autophagosome that delivers the Ag85B-chaperone complex to lysosomes for autophago-lysosomal degradation and p25-epitope presentation to CD4 T cells. Interestingly, blockade of proteasomes led to an increase in CHIP activity of DCs enhancing p25-epitope presentation to CD4 T cells. Thus, intrinsic degradation of subunit vaccines through proteasomes may prevent the efficacy of MHC-II pathway while CHIP dependent autophagy may provide a means to enhance epitopes for CD4 T cells.
Interferon-γ receptor and STAT1 signaling in B cells are central to spontaneous germinal center formation and autoimmunity
Authors: Domeier Phillip P , Chodisetti Sathi Babu , Soni Chetna , Schell Stephanie L , Elias Melinda J , Kitamura Daisuke , Rahman Ziaur
Published: January 2023

In Systemic Lupus Erythematosus (SLE), spontaneously developed germinal centers (Spt-GCs) harbor AutoAb-producing B cells that promote disease, but the mechanisms that control Spt-GC development are not clear. Here we report that B cell-intrinsic IFNγR and STAT1 signaling are essential for Spt-GC formation. The IFNγR deficiency, however, does not significantly affect foreign Ag-induced GC and Tfh responses, suggesting that two distinct mechanisms regulate foreign-Ag vs Spt-GC responses. We also demonstrate that IFNγR-mediated STAT1 signaling drives T-bet expression, pro-GC gene expression and IFNγ production by B cells, which are critical for Spt-GC and Tfh development. To understand how IFNγ signaling regulates the Spt-GC response and associated AutoAb production in SLE, we utilized B6.Sle1b mice that harbor the lupus-prone NZM2410 strain-derived SLAM locus. B6.Sle1b mice exhibit elevated Spt-GC and follicular helper T cell (Tfh) responses that associate with impaired B cell selection in GCs. B6.Sle1b mice with B cell intrinsic IFNγR deficiency have significantly reduced Spt-GC and Tfh responses resulting in a markedly lower number of IgG-producing antibody forming cells and diminished IgG2c and IgG2b autoantibody titers compared to B6.Sle1b mice. Additionally, the proliferation and differentiation of DNA-reactive B cells into a GC B cell phenotype require B cell-intrinsic IFNγR signaling. Together, our data define a novel B cell-intrinsic IFNγR signaling pathway in Spt-GC development and autoimmunity.
Stability of Foxp3 mRNA is controlled by SAMHD1 in human T regulatory cells
Authors: Kim Yong Chan , Kim Kee Kwang , Yoon Jeong Heon , Shevach Ethan M , Scott David W
Published: January 2023

Clinical application of antigen-specific T regulatory cells (Tregs) offers promise for the treatment of undesirable immune diseases. To achieve this goal, long-term expansion of Tregs is required to obtain sufficient numbers of cells. However, human Tregs are not stable ex vivo. Therefore, we previously developed an innovative Treg expansion protocol using phosphorothioated random oligonucleotides (ODNps25). The addition of ODNps25 successfully resulted in the stabilization of engineered antigen-specific Tregs, however, the mechanism is not fully characterized. We first identified SAMHD1 (sterile alpha motif and HD domain 1) as an ODNps25 binding protein using a UV-crosslinking pull-down strategy. SAMHD1 physically interacted with the 3′ untranslated region (UTR) of Foxp3 mRNA and was translocated from nucleus to cytoplasm after ODNps25 treatment. Importantly, addition of ODNps25 enhanced the interaction of SAMHD1 and Foxp3 mRNA significantly, and this interaction was increased by TCR stimulation. Since ODNps25 binds to the nuclease (HD) domain of SAMHD1, we then established that overexpression of a nuclease-deficient mutant (D137N) in Tregs significantly stabilized the expression level of Foxp3 protein. This mutant also enhanced stability of a luciferase reporter, followed by the 3′ UTR of Foxp3 mRNA. Additionally, we also found that phosphorylation of the threonine residue (Thr592), which is a regulatory site to control SAMHD1 activity, was clearly downregulated in Tregs. Taken together, we suggest that ODNs interact with the HD of SAMHD1 to interfere with its nuclease activity, thereby stabilizing 3′ UTR of FoxP3 mRNA in long-term culture.
Enhanced PI3K/AKT/mTOR responses are associated with resistance to activation-induced cell death and expansion of memory CD4+ T cells in T1D subjects and healthy carriers of the PTPN22 risk variant
Authors: Habib Tania J , Turner Michael S , Anderson Warren , Funk Andrew , Rieck Mary , Puri Kamal D , Greenbaum Carla , Long S. Alice , Buckner Jane H.
Published: January 2023

The genetic variant PTPN22 1858T, encoding Lyp620W, confers increased risk for multiple autoimmune diseases including Type 1 diabetes (T1D). To date, Lyp620W has been associated with increased CD4+memory T cells and enhanced Th1 responses in healthy carriers. However, it is not clear whether this is due to altered lineage commitment and/or survival within the CD4+memory T cell compartment. Here, we determined the mechanism by which Lyp620W alters human CD4+memory T cell homeostasis in PTPN22 1858T carriers and T1D subjects. T1D subjects displayed significantly increased CD4+ memory T cells as compared to healthy individuals irrespective of PTPN22 genotype, which was further increased in homozygous carriers of PTPN22 1858T, suggesting a partial role for this variant. Among healthy 1858T carriers, we found a significant increase in the magnitude of Th1 effector responses, but not enhanced Th1 differentiation. Consistent with increased survival, TCR-induced PI3K/AKT/mTOR pathway responses and BCL-2 expression were augmented in CD4+ memory T cells from healthy 1858T carriers. Importantly, these alterations were associated with increased resistance to TCR-mediated apoptosis in CD4+ memory T cells. Similar changes in PI3K/AKT signaling and T cell survival were observed in the Th1 memory compartment of T1D individuals, and selective inhibition of PI3Kδ reversed the survival phenotypes in memory T cells from both healthy 1858T carriers and T1D subjects. Thus, our combined findings reveal a novel immune ‘signature’ associated with Lyp620W and highlight a clinically-relevant T cell intrinsic mechanism that promotes the survival of potentially pathogenic Th1 effector T cells in T1D.
Epigenetic modulation of inflammatory T cell responses
Authors: Webb Lindsay M , Savardekar Himanshu , Jablonski Kyle , Amici Stephanie A , Panfil Amanda , Smith Porsha , Green Patrick , Li Chenglong , Baiocchi Robert A , Guerau-De-Arellano Mireia
Published: January 2023

Pro-inflammatory T helper (Th) 1 and Th17 cell responses drive autoimmune attack of the central nervous system in Multiple Sclerosis (MS), resulting in disability. In contrast, regulatory Th2 and Treg responses are deficient. The reactivation and expansion of inflammatory T cells is linked to increased severity and active MS disease. Thus, therapeutic strategies that modulate this process would be beneficial. Epigenetic modifications such as histone methylation and acetylation provide control over cellular phenotype. The epigenetic modifying enzyme Protein arginine methyltransferase 5 (PRMT5) is the only known enzyme known to catalyze symmetric dimethylation (SDM) on arginine residues of histones. Although PRMT5 has been shown to be up-regulated in several cancers, there is currently no known role for PRMT5 in T cells. We have found that PRMT5 was up-regulated during re-activation of myelin-specific mouse memory T cells and Experimental Autoimmune Encephalomyelitis (EAE), suggesting that PRMT5 may control expansion of pathogenic autoimmune responses. To test this, we developed novel PRMT5-selective inhibitors in collaboration with the Drug Development Institute at OSU. These novel PRMT5 inhibitors preferentially suppressed Th1 over Th2 cell proliferation, an effect that was partially dependent on IL-2 suppression. In addition, we observed similar effects in human memory T cells, suggesting a conserved function of PRMT5 in both human and mouse. Genetic knockdown of PRMT5 with a short hairpin RNA similarly suppressed inflammatory Th1 cell proliferation. Overall, these data support a role for PRMT5 in memory T cell responses and as a promising therapeutic target in autoimmunity.
Mincle activation and the Syk/Card9 signaling axis are central to development of autoimmune disease of the eye
Authors: Lee Ellen , Brown Brieanna , Snow Paige , Vance Emily , Silver Phyllis , Wells Christine , Caspi Rachel R , Rosenzweig Holly L.
Published: January 2023

We recently identified the innate receptor Mincle, a C-type lectin receptor (CLR) that signals through Syk and Card9, as essential in induction of ocular autoimmunity, using experimental autoimmune uveitis (EAU). Now, we further delineate molecular and cellular factors involved in orchestrating this Mincle-dependent mechanism. EAU is typically induced by immunization with interphotoreceptor retinoid-binding protein (IRBP) in CFA and then evaluated by fundus imaging and histology. When the synthetic Mincle agonist TDB (trehalose-6-6′-dibehenate) was used in lieu of CFA, TDB-immunized mice developed uveitis (p<0.05; n=8/group). This effect required Mincle and its downstream signaling adaptor, Card9, as EAU was reduced in the absence of either molecule (p<0.05, n=10–15/group). When Syk, a kinase upstream of Card9, was pharmacologically inhibited by piceatannol (given i.p., q4d, d0–16), TDB-induced EAU was abrogated (p<0.05 vs. control; n=15/group). These results demonstrated that the adjuvant effect of innate signaling through Mincle is necessary and sufficient to support EAU. Studies with bone marrow (BM) chimeras, performed to define the cellular source of Card9 and Mincle-activated responses, showed that Card9 expression was required in hematopoietic, rather than non-hematopoietic cells (p<0.05; n=10/group). Since Card9 is primarily expressed by myeloid cells, Cre-Lox recombination was used to generate conditional KO mice with CD11c+ dendritic cells (DC) lacking Syk. Syk-deficiency in DCs resulted in reduced disease early after EAU induction (p<0.05 vs. control; n=6–10/group). These studies uncover an essential role for Mincle and the Syk/Card9 axis in autoimmune uveitis.
Deficiency of protein tyrosine phosphatase sigma abolishes resistance of BALB/c mice to EAE by modulating dendritic cell function
Authors: Kong Weimin , Ohtake Yosuke , Li Shuxin , Ganea Doina
Published: January 2023

Recent studies reported that protein tyrosine phosphatase sigma (PTPσ) is expressed in plasmacytoid dendritic cells and that PTPσ−/− mice are more susceptible to inflammatory bowel disease. However, the function of PTPσ in autoimmune diseases has not been explored. The present study is the first report on the effects of PTPσ in experimental autoimmune encephalomyelitis (EAE). In contrast to wild type BALB/c mice which are resistant to EAE, PTPσ−/−BALB/c mice developed EAE. Pathohistology studies indicated severe demyelination, axonal loss and increased immune cell infiltration in the spinal cords of PTPσ−/− BALB/c mice. Gene expression analysis of spinal cords showed increased expression of proinflammatory cytokines and chemokines in PTPσ−/− BALB/c mice. Ex vivo experiments showed higher levels of IFNg and IL17 production in splenocytes isolated from PTPσ−/− EAE mice. PTPσ was highly expressed in conventional dendritic cells (cDC) but not in CD4+ T cells. As a result of PTPσ deficiency, cDC exhibited an inflammatory phenotype characterized by enhanced expression of pro-inflammatory cytokines, chemokine receptors and migration capacity, and decreased expression of anti-inflammatory cytokines. Furthermore, adoptive transfer of PTPσ−/− DC induced more severe disease compared to wild type DC. In conclusion, we established that PTPσ is a negative regulator in EAE, and that this is mediated, at least partially, through effects on DC function.
Heightened preclinical dysregulation of distinct adaptive and renal-associated mediators in patients who develop nephritis as they transition to systemic lupus erythematosus classification
Authors: Munroe Melissa , Anderson Jourdan R , Robertson Julie M , Niewold Timothy B , Tsokos George C , Keith Michael P , Merrill Joan T , Buyon Jill P , Harley John B , James Judith A
Published: January 2023

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease marked by immune dysregulation. Why some patients have only moderate symptoms and others develop organ-threatening manifestations is unclear. This study evaluates the temporal expression of autoantibodies and cytokines in sera from the Department of Defense Serum Repository during transition from preclinical lupus to SLE in patients (n=83) who do or do not present with nephritis. Patients who met renal criteria (n=30; 36%) did so within 5.2 (±5.5) months of SLE classification (range −5.2 to +12 months). Renal cases experienced earlier onset of autoantibody positivity vs. non-renal cases (mean −3.8 vs −2.6 years relative to SLE classification, p=0.0442). Prior to developing nephritis, renal cases exhibited elevated levels of distinct soluble mediators compared to matched non-renal cases and healthy controls, including adaptive mediators IL-4, IL-5, IL-12, and IFN-γ, chemokines IP-10 and MCP-3, and nephritis-associated mediators SCF and shed TNFRII (all p<0.05), increasing again at the time of nephritis (all p<0.02 compared to pre-nephritis levels). These same mediators were significantly increased at SLE classification in patients with nephritis vs. non-renal patients (all p<0.0001). These data indicate that perturbations in distinct immune mediated inflammatory processes may help identify individuals at high risk of renal involvement for early and continued monitoring and intervention.
Searching for citrullinated autoantigen biomarkers for interstitial lung disease associated with Rheumatoid Arthritis
Authors: Ganesan Vinitha , Harlow Lisa , Kaku Barclay Machika , Gigilio Marisa , Ascherman Dana P , Minden Jonathan S
Published: January 2023

Interstitial lung disease (ILD) associated with Rheumatoid arthritis (RA) is the cause of death in ~13% of RA patients. Biomarkers are therefore urgently needed to predict ILD development in RA patients, so that appropriate treatment can be instituted. Citrullination occurs in the lungs of RA-ILD patients. We hypothesize that, this post-translational modification leads to the formation of neo-epitopes, triggering autoreactivity & inflammation of the lungs. Because the formation of citrullinated autoantigens and associated autoantibodies is likely linked to ILD in RA patients, we are using RA & RA-ILD patient sera to differentially immunoprecipitate (IP) in-vitro citrullinated HeLa protein extracts containing potential citrullinated autoantigens. When using mass spectrometry (MS) for protein identification, IP results are confounded by an overwhelming amount of antibody-derived peptides. To solve this, we developed a secondary purification step using Biotin-CDM, a novel reversible protein capture reagent that tags all proteins in a cell lysate. The captured biotinylated IP proteins are then separated from non-biotinylated antibodies and released in a un-biotinylated state in the appropriate buffer for proteomic analysis, free of contaminating antibodies. With this modified approach, we have detected several differences in citrullinated autoantigen recognition profiles between RA & RA-ILD patient sera. We are currently identifying these differential IP proteins using MS, with the goal of using target antigens as “bait” in co-IP to isolate other interacting proteins from lung tissue extracts. Once validated, these results will help us further our understanding of the complex networks involved in RA-ILD pathogenesis.
Immunobiology of antigen presenting cells during relapse vs. remission in multiple sclerosis
Authors: Sinha Sushmita , Cunnusamy Khrishen , Chaudhary Parul , Greenberg Benjamin M , Frohman Elliot M , Karandikar Nitin J
Published: January 2023

Multiple sclerosis (MS) is the most common cause of neurological disability in young adults. Approximately 80% of MS patients initially present with the relapsing-remitting disease. Although studies suggest immune hyperactivity during relapse, role of specific immune cells has been understudied. We investigated whether APCs might be the drivers of inflammation during an MS-relapse. Paired blood samples were obtained from patients during relapse (Rel) and remission (Rem) phase of MS. APCs were immunophenotyped for activation, inhibition and migration-associated markers. T cell-cytokine stimulatory potential of Rel vs. Rem-APCs was studied by co-culturing sorted APC-subtypes with healthy donor-CD3+ T cells. Rel-monocytes displayed a significantly higher expression of adhesion molecules while inhibitory receptor ILT4 was significantly increased on Rem-monocytes. Further, Rel-monocytes stimulated significantly more IFN-g and IL-17 from allogeneic CD4+ T-cells. Rel- B-cells had a significantly higher expression of adhesion molecules and displayed significantly increased potential of stimulating IFN-g from allogeneic CD8+ T-cells. Inhibitory receptor ILT3 was significantly increased on PDCs during Rem. Functionally, Rel-MDC stimulated significantly more IL-17 and IFN-g from allogeneic CD4+ and CD8+ T-cells, respectively. Overall, our study suggests that these key differences in APC subsets during Rel vs Rem phases could potentially modulate T cell responses by favoring more pro-inflammatory T-cell phenotype during MS relapse. Combined with our publications related to differential regulatory ability of CD4 and CD8 T-cells this study provides great insights into the underlying immunopathology of an MS relapse.
Emerging roles of IFNα, IL-10, IL-1 and IL-8 in central nervous inflammation and immune response imbalance in transgenic porcine model of neurodegenerative Huntington’s disease
Authors: Kovarova Hana , Jarkovska Karla , Valekova Ivona , Kotrcova Eva , Juhas Stefan , Motlik Jan , Bucci John , Gadher Suresh Jivan
Published: January 2023

Huntington’s disease (HD) is an inherited neurodegenerative disease with impairment of motor and cognitive functions. Studies in HD gene carriers expressing mutant huntingtin protein (mHTT) demonstrated altered immune response indicating that cytokines may have a significant role in disease development.

We utilized the novel transgenic HD model bearing N-fragment of human mHTT in minipig (Baxa M et al., J Huntington’s Dis. 2013, 2:47–68), to investigate central nervous and peripheral cytokine levels. Using Luminex® xMAP™ multiplexing technology and Thermo Fisher Scientific Inc., Swine Cytokine Magnetic 7-plex Panel (Invitrogen™ LSC0001M), the biological fluids were interrogated to identify cytokine alterations related to HD progression. The modelling of interaction networks between such cytokines and HTT facilitated study of intracellular signaling pathways associated with immune response dysregulation.

The most pronounced change was the decline of IFNα in cerebrospinal fluid and secretome of microglial cells (MG). In addition, IL-10 was also lower in the same biological fluids. Elevated levels of pro-inflammatory IL-1 and IL-8 were secreted by MG whilst IL-8 was also increased in serum of transgenic minipigs. The high proportion of mHTT in MG may have a causative impact on cytokine production. A novel role for CREB-binding protein in HD pathogenesis was highlighted.

These findings underlined the roles of IFNα, IL-10, IL-1 and IL-8 in central nervous system inflammation and immune response imbalance in HD progression warrants further investigation into their usefulness in human patients.

Acknowledgement

We thank CHDI, MESY Projects EXAM; CZ.1.05/2.1.00/03.0124, LO1609 and Thermo Fisher Scientific for expertise.
Fractalkine signaling during systemic endotoxemia inhibits perivascular microglial lesion formation in the diabetic retina
Authors: Mendiola Andrew S , Garza Rolando , Cardona Sandra M , Cardona Astrid E
Published: January 2023

It’s known that diabetics are more susceptible to infections and there is an association between diabetes and chronic inflammation, but whether systemic inflammation contributes to retinopathy remains unknown. Microglial-mediated inflammation has gained recognition as a key contributor to diabetic retinopathy (DR) pathogenesis. We have shown that signaling between the chemokine fractalkine (FKN) and CX3CR1 on microglia is important to maintain neuroprotection and that CX3CR1-deficiency directs microglial-mediated inflammation and neurotoxicity in the diabetic retina. To extend these studies, we tested the hypothesis that acute endotoxemia perpetuates microglial activation and that CX3CR1-deficent mice will be more susceptible to retinal pathology due to dysregulated microglial responses. Systemic endotoxemia was induced in nondiabetic and diabetic CX3CR1-HET and CX3CR1-KO mice by administration of four-daily injections of LPS (1 mg/kg/mouse; i.p.; n=12). Confocal analysis of retinal tissue revealed that in CX3CR1-KO mice, endotoxemia induced a robust-cellular activation represented by morphological changes, IL-1β, iNOS expression, and intense perivascular clusters of microglia. This phenotype was exacerbated in the diabetic CX3CR1-KO retina. These microglial lesions observed in CX3CR1-KO mice coincided with perivascular accumulation of the blood-protein fibrinogen. Lastly, fractalkine treatment (intraocular; 30 ng) into FKN-KO mice dampened microglial activation and astrogliosis during acute endotoxemia. These data suggest that systemic inflammation influences breakdown of the blood-retinal barrier, and may provide therapeutic advances by fractalkine treatment to mitigate DR pathogenesis.
Investigating the Role of IL-33 in the Pathogenesis of Familial Mediterranean Fever
Authors: Keskin Goksal , tutluer Seda , Inal Ali , Cerci Pamir , Olmez Umit
Published: January 2023

Familial mediterranean fever (FMF) is an autosomal recessive genetic disorder familial disease with inflammatory reactions of serosal membranes characterized by recurrent episodes of abdominal chest and joint pain, and fever. During the acute attacts, increased several proinflammatory cytokines have been described. IL-33 is a member of the IL-1 cytokine superfamily. It has been reported that IL-33 plays an important role in inflammation and homeostasis. Here, we analyzed serum IL-33 concentration in patients with FMF to assess its possible role in the pathophysiology of this disease.

Material and method

In this study, 58 patients with FMF (21 female, 37 male; mean age 27.8 ± 5.2 years, mean disease duration 8.2 ± 3,7 years) and 22 healthy controls (10 female, 12 male, mean age 26,5 ± 5,9 years) were included. Serum IL-33 levels were measured by ELISA.

Results

The mean serum IL-33 levels were 3,88 ± 2,25 pg/ml in patients with FMF and 2,89 ± 0.48 pg/ml in the healthy controls. The mean levels of serum IL-33 were 5,48 ± 2,13 pg/ml in active stage and 2,27 ± 0,36 pg/ml in inactive stage. According to these results; serum IL-33 levels were significantly higher in patients with FMF compared with healthy controls (p<0.01). In addition, Serum IL-33 levels were significantly higher in active stage compared to in inactive stages and controls (p<0.0001 and p<0.0001). In the inactive patients with FMF, serum IL-33 concentrations were found to be lower compared to healthy controls (p<0.05). In active FMF patients, the mean serum IL-33 level was correlated with arthritis (p<0.05).

Conclusion

The high levels of serum IL-33, in active patients with FMF suggest that IL-33 may play a significant role of in the pathogenesis of FMF.
Expression of CCR5 and its ligand CCL5 in pancreatic cancer
Authors: Singh Santosh Kumar , Banerjee Saswati , Lillard James W. , Singh Rajesh
Published: January 2023

Despite recent advancements in understanding of pancreatic cancer (PC) molecular features and their impact on clinical behavior, PC systemic therapy decision-making is still guided by a constellation of clinico-pathological features, which are derived from histo-pathological analysis of primary PC tissues. Chemokines and their corresponding receptors play a crucial role in metastasis and organ specific homing of cancer cells in different cancers including PC. CCL5 is a homeostatic chemokine acts through its cognate receptor, CCR5. Here, we have provided the evidence that CCR5 and CCL5 are significantly elevated in both PC cell lines (BxPC-3, MIA PaCa-2 and AsPC-1) and clinical samples. Flow cytometric analysis of PC cell lines (BxPC-3, MIA PaCa-2 and AsPC-1) show significantly higher expression of CCR5. These findings were further confirmed at mRNA expression level by real-time PCR analysis. To determine the clinical significance of CCR5 expressed by PC cell lines, pancreatic cancer tissue microarray was stained for CCR5 and CCL5. Aperio scan scope scanning system was used to numerically analyze immuno-histochemical staining of CCR5 and CCL5. Our results show significantly (p < 0.001) higher expression of CCR5 and CCL5 in cancerous tissues compared to normal pancreatic tissues. These data demonstrate both biological and clinical significance of CCR5 and CCL5 expression that may contribute to PC metastasis.
Human skin mast cells constitutively express gp130 and functional IL-6 receptor
Authors: McHale Cody C , Deppen Juline , Shirley Devon , Gomez Gregorio
Published: January 2023

Mast cells, the effector cell type of allergic reactions, have been implicated in numerous traditionally non-allergic diseases including cancer, diabetes, and obesity. Interleukin-6 (IL-6) is a pleiotropic cytokine involved in many of these pathologies suggesting that IL-6 could be the link that connects mast cells to these disease states. However, it had not been formally demonstrated that human primary mast cells express a functional IL-6 surface receptor. In this study, we show that human skin mast cells (hSMCs) constitutively express IL-6Ra and gp130, a common signal transducing chain shared by members of the IL-6 cytokine family that includes leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, IL-11, cardiotrophin 1, IL-35 and IL-27. Whereas previous studies with mouse bone marrow-derived mast cells showed that IL-10 was required for surface expression of gp130, we found that IL-10 did not enhance expression on hSMCs although gp130 mRNA was increased. IL-6R functionality is demonstrated by the IL-6-induced phosphorylation of STAT3, and increased expression of SOCS3 mRNA. Interestingly, IL-6 gene expression was enhanced with IL-6 itself suggesting a positive feedback mechanism. Similar results were obtained with IL-27, which also utilizes gp130 together with WSX-1 for signaling. Mast cell degranulation was not induced with IL-6 or IL-27 alone, and neither cytokine affected FcɛRI-induced release of β-hexoaminidase. However, IgE-dependent TNF production was enhanced with IL-27. These data demonstrate that human mast cells can be directly activated by IL-6 or other gp130-utilizing cytokines, and support the notion that IL-6 could be a link that connects mast cells to non-allergic disease.
Differential Expression of the Transcription Factor SPI1 (PU.1) in Dendritic Cell Subsets
Authors: Soper David M , Madeux Danny , Yang Xifeng
Published: January 2023

SPI1 belongs to the E26-transformation-specific (Ets) family of transcription factors and is exclusively expressed in hematopoietic cells. Originally identified as an oncogene that blocks erythroblast differentiation, SPI1 plays a critical role in the development and lineage commitment of both myeloid and lymphoid cells. Gene knockout studies show that spi1−/− mice exhibit a multi-lineage defect in the generation of monocytes, granulocytes, B, T, and dendritic cells. While SPI1 expression is required for normal hematopoietic cell development, the level of SPI1 expression influences specific lineage commitment. Using in-house generated species-specific clones, human peripheral blood mononuclear cells (PMBCs) and mouse splenocytes were evaluated for SPI1 expression by flow cytometric analysis. In human PMBCs, Lin− HLA-DR+ CD11c+ CD303− monocyte-derived dendritic cells (mDCs) exhibit high levels of expression while Lin− HLA-DR+ CD11cint CD303+ plasmacytoid dendritic cells (pDCs) do not express SPI1 protein. These data suggest SPI1 expression is required for the development and function of human mDCs, while SPI1 expression is not necessary for terminally differentiated human pDCs. Interestingly, SPI1 expression is detected in both murine DC subsets: Lin− I-A/I-E+ CD11c+ CD317− mDCs and Lin− I-A/I-E+ CD11cint CD317+ pDCs, although the expression level in pDCs is significantly lower than in mDCs. These observations indicate expression patterns differ between mouse and human cells, and demonstrate that DC subsets exhibit differential expression of SPI1 protein.
Zeb2 is required for development of plasmacytoid dendritic cells
Authors: Wu Xiaodi , Briseño Carlos G. , Grajales-Reyes Gary E. , Wumesh KC , Kretzer Nicole M. , Murphy Theresa L. , Murphy Kenneth M.
Published: January 2023

Recent work has shown that the transcription factor Zeb2 is regulated by and cooperates with T-bet to promote terminal differentiation of NK cells and cytotoxic T cells. In the mouse, Zeb2 is expressed throughout dendritic cell (DC) development and in mature plasmacytoid DCs (pDCs) and CD11b+ classical DCs (cDCs), but not in CD8+/CD103+ cDCs. We hypothesized that downregulation of Zeb2 expression would be essential for the development of CD8+/CD103+ cDCs. Upon conditional deletion of Zeb2, we observed a substantial decrease in pDC frequency and an increased ratio of CD8+:CD11b+ cDCs; meanwhile, Zeb2 haploinsufficiency yielded a partial defect in pDC development. Additional experiments demonstrated that this defect was cell-intrinsic and durable, and that it could not be rescued by administration of type I interferon, which increased pDC frequency in cultures of wild-type DC progenitors but directed Zeb2-deficient DC progenitors almost entirely to the CD8+/CD103+ cDC lineage. Deletion of Zeb2 induced by poly(I:C) treatment in an Mx1-Cre-driven model also resulted in perturbations in other myeloid lineages. Although poly(I:C) treatment led to an acute loss of Ly-6Chi monocytes both in Zeb2fl/fl;Mx1-Cre(tg) mice and in control mice, those monocytes were replenished to baseline frequencies in control mice but not in Zeb2-deficient mice. Similar decreases in monocyte frequency were observed in other organs, and further experiments suggested that this defect was also cell-intrinsic, gene dosage-dependent, durable, and observable in model systems that do not require use of poly(I:C). Thus, we have identified an essential regulator of development in multiple myeloid lineages.
IRF4 and IRF8 act in CD11c+ cells to regulate terminal differentiation of lung tissue dendritic cells
Authors: Kovats Susan , Turner Sean , Paul Jinny , Ainsua-Enrich Erola , Bajana Sandra
Published: January 2023

Dendritic cells (DCs) initiate immune responses in barrier tissues including lung and skin. Conventional DC subsets, CD11b−(cDC1s) or CD11b+(cDC2s), arise via distinct networks of transcription factors involving IRF4 and IRF8 and are specialized for unique functional responses. Using mice in which a conditional Irf4 or Irf8 allele is deleted in CD11c+ cells, we determined if IRF4 or IRF8 deficiency beginning in CD11c+ cDC precursors (pre-cDCs) changed the homeostasis of mature DCs or pre-DCs in the lung, dermis and spleen. CD11c-cre-Irf4−/− mice selectively lacked a lung-resident CD11chiCD11b+SIRPa+CD24+ DC subset, but not other lung CD11b+DCs or alveolar macrophages. Numbers of CD11b+CD4+ splenic DCs, but not CD11b+ dermal DCs, were reduced, indicating cDC2s in the lung and dermis develop via different pathways. Irf4 deficiency did not alter numbers of cDC1s. CD11c-cre-Irf8−/− mice lacked lung-resident CD103+ DCs and splenic CD8a+ DCs, yet harbored increased IRF4-dependent DCs. This correlated with a reduced number of Irf8−/− pre-cDCs, which contained elevated IRF4, suggesting that Irf8 deficiency diverts pre-cDC fate. Analyses of Irf4 and Irf8 haploinsufficient mice showed that while one Irf4 allele was sufficient for lung cDC2 development, two functional Irf8 alleles were required for differentiation of lung cDC1s. Thus, IRF8 and IRF4 act in pre-cDCs to direct the terminal differentiation of cDC1 and cDC2 subsets in the lung and spleen. These data suggest that variation in IRF4 or IRF8 levels resulting from genetic polymorphisms or environmental cues will govern tissue DC numbers and therefore regulate the magnitude of DC functional responses.
IL-17A is rapidly triggered by staphylococcal superantigens and is pathogenic in toxic shock syndrome
Authors: Szabo Peter Anthony , Goswami Ankur , Welch Ian , McCormick John , Haeryfar S.M. Mansour
Published: January 2023

Toxic shock syndrome (TSS) is a life-threatening illness characterized by high-grade fever, erythematous rash, hypotensive shock, and multi-organ dysfunction within hours of onset. It is caused by exposure to bacterial exotoxins known as superantigens (SAgs), which are produced by the common bacterium Staphylococcus aureus. During infection, SAgs activate up to 20% of T cells inducing a severe systemic inflammatory response that can lead to vascular leakage, multiple organ damage and death. However, the role of IL-17A, a potent inflammatory cytokine, in this potentially fatal reaction remains virtually unexplored. We have found that human peripheral blood mononuclear cells (PBMCs) stimulated with SAgs in vitro immediately up-regulate IL-17A mRNA (up to 12,000-fold) and produce substantial amounts of IL-17A protein within hours. Utilizing the cutting-edge RNA-Flow Cytometry technique, we identified a subset of memory T cells that was responsible for the rapid IL-17A response. We also investigated the effect of IL-17A signaling on downstream inflammatory cytokine and chemokine gene expression via IL-17A receptor blockade. Lastly, we used HLA-DR4 transgenic mice to demonstrate rapid IL-17A production in response to SAgs, similar to human PBMCs. Neutralizing IL-17A in this model greatly reduced tissue damage, morbidity and mortality. Taken together, our results reveal a novel role for memory T cells in TSS and define a previously unrecognized contribution by IL-17A to the initiation and pathogenesis of the syndrome.
An antagonistic role for Fyn and Lyn kinases in autoimmune arthritis
Authors: Paranjape Anuya R , Ryan John J
Published: January 2023

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder characterized by infiltration of innate immune cells into the synovial joints, whose etiology is unclear. While the Fcγ receptor (FcγR) aggregation on innate inflammatory cells by auto-antibodies is a critical stimulus, much of FcγR signaling remains unknown. Previous research from our lab has shown that FcγR-mediated innate immune cell activation is antagonistically controlled by the Src family kinases, Fyn and Lyn. Our current study investigates the Fyn/Lyn dichotomy in controlling the severity of the inflammatory arthritis using K/BxN model or RA. Our preliminary studies show that Lyn deletion exacerbates joint swelling and clinical arthritis scores, while Fyn deletion reduces plasma inflammatory cytokine levels. These data indicate an antagonistic role for Fyn and Lyn kinases in autoimmune arthritis and support further study of the mechanisms by which these Src family kinases regulate inflammation.