bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of baseline cytokine levels with anti-tumour necrosis factor treatment response in rheumatoid arthritis
Authors: Gibson David S , McGeough Cathy M , Slevin Mary M , Wright Gary , Gardiner Philip , Murray Helena A , Latten Mark J , Crockard Martin A , Lamont John V , Bjourson Anthony J
Published: January 2023

Background

Clinical trials of anti-tumour necrosis factor alpha (TNF) have shown efficacy in 60–70% of rheumatoid arthritis patients (RA). Predicting response to anti-TNF drugs at baseline remains an elusive goal in RA management. The purpose of this study was to determine if baseline levels of circulating cytokines, soluble receptors, adhesion molecules and metabolic factors differentiate future responders.

Methods

RA patients (n=29) with active disease were recruited, who had failed on disease modifying drugs and were recommended for anti-TNF treatment. Peripheral blood samples were collected at baseline. Responders were identified by a ≥1.2 reduction in disease activity score (DAS28-ESR) at 6 months. Five protein arrays quantified 33 proteins in pre-treatment plasma (Cytokine I, III and IV; Metabolic I; Adhesion molecule; Randox Laboratories Ltd., UK). Data was analysed by Pearson ranked correlation and logistic regression to clinical measures of disease activity at baseline and six months anti-TNF treatment.

Results

Elevated levels of interleukin 8 (p=0.043), monocyte chemoattractant protein (p=0.027), granulocyte-macrophage colony-stimulating factor (p=0.020) and soluble interleukin 2α receptor (p=0.01) were associated with high TJC at baseline. No significant relationship with the protein array panels was recorded relative to baseline DAS28-ESR. Significant inverse correlations were observed between absolute change in DAS28-ESR after 6 months of anti-TNF therapy and baseline levels of interleukin 6 (p=0.026) and resistin (p=0.044).

Conclusions

These findings suggest that baseline levels of specific circulating proteins may help to differentiate RA patient responders to anti-TNF therapies.
Catalase expression mediates redox regulation of autophagy and promiscuous gene expression in thymic stromal cells.
Authors: Hester Allison K , Semwal Manpreet Kaur , Xiao Yangming , Cepeda Sergio , Venables Thomas , Griffith Ann V
Published: January 2023

T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells (TSCs) directs progenitors along a well-characterized differentiation program. However, the biology of stromal cells comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the peroxide quenching enzyme catalase (CAT) in thymic stromal cells, and found that CAT deficiency results in high reactive oxygen species (ROS) levels in this population, eventually leading to thymic atrophy in aged mice. Here, we find that when catalase deficiency is complemented by overexpression targeted to mitochondria in transgenic mice (mCAT Tg), both ROS levels and stromal function decline in young mice relative to non-transgenic littermates. TSC transcriptome analysis reveals decreased expression of tissue-restricted antigen (TRA) and autophagy pathway genes in Cat Tg mice. Analysis of autophagy flux reporter mice ubiquitously expressing the RFP-EGFPLC3 fusion transgene also indicates diminished autophagic flux in mCAT Tg mice, particularly in TSCs. Stromal TRA expression and autophagic flux are required for self-antigen presentation, and therefore promote negative selection of potentially auto-reactive T cells. We propose that oxidative stress generated by low catalase levels in stromal cells promotes these key physiological functions in the young, steady state thymus; in contrast however, the resulting accumulated oxidative damage ultimately impairs function in the aged thymus.
Requirement for PBX1 in developmental programming of natural killer cells
Authors: Yonggang Zhou , Binqing Fu , Xiuxiu Xu , Xianhong Tong , Rui Sun , Zhigang Tian , Haiming Wei
Published: January 2023

Maintenance of the optimally functional natural killer (NK) cells pool is essential for the host immune surveillance. Some intracellular growth-promoting factors (GPFs) have been reported as a vital new molecular component for maintenance homeostatic expansion of NK cells, but transcriptional regulation mechanism of Gpfs genes in NK cells is unclear. To better understand the underlying mechanism for NK cells be regulated to express GPFs as a secreted or intracellular isoform, we investigated the potential role of transcription factor PBX1 within the NK cell compartment by using Pbx1 gene-targeted mice.

In terms of NK cell late development and homeostatic proliferation, here we report that PBX1 is the key transcriptional factor that underpins both of these events. The transcriptional factor PBX1 and GPFs are expressed in NK cell precursors and immature NK cells. After Pbx1 is knocked down on NK cells, NK cells shown a serious decline in the ability of expressing these GPFs. PBX1 regulates the transcription of GPFs genes in NK cells via direct binding. In the NK cell development, knocked down or excision of Pbx1 in mice harbor reduction of NK cells and the Pbx1-deficient developmental phenotype is NK cell intrinsic. In the NK cell homeostasis, PBX1 is necessary to support NK cell homeostasis in BM or spleen niche. The Pbx1-deficient mice is accompanied by impaired transition of NK cells from immature to mature, and diminished ability to differentiate into memory-like NK cells due to the impairment of GPFs.

Our study indicates that PBX1 represent a novel regulatory element for supporting the homeostatic developmental program of NK cells, and provides the basis for improved approaches to immunotherapy for virus clearance and tumor surveillance.
Alterations in the thymic selection threshold skew the self-reactivity of the T cell receptor repertoire in neonates
Authors: Dong Mengqi , Artusa Patricio , Kelly Stephanie A. , Fournier Marilaine , Baldwin Troy A. , Mandl Judith N. , Melichar Heather J.
Published: January 2023

Neonatal and adult T cells differ in their effector functions. Although it is known that cell-intrinsic differences in mature T cells contribute to this phenomenon, the factors involved remain unclear. Given emerging evidence that the binding strength of the TCR for self-peptide presented by MHC (self-pMHC) impacts T cell function, we sought to determine if altered thymic selection thresholds influence the self-reactivity of the TCR repertoire during ontogeny. We found that conventional and regulatory T cell subsets in the thymus of neonates and young mice expressed higher levels of cell-surface CD5, a surrogate marker for TCR avidity for self-pMHC, as compared to their adult counterparts and that this difference in self-reactivity was independent of their distinct TCR repertoire. The increased binding strength of the TCR repertoire for self-pMHC in neonates was not solely due to reported defects in clonal deletion. Rather, our data suggest that thymic selection is altered in young mice such that thymocytes bearing TCRs with low affinity for self-peptide are not efficiently selected into the neonatal repertoire and stronger TCR signals accompany both positive and agonist selection. Importantly, the differences in CD5 levels reflect physiologically relevant differences based on the preferential expansion of T cells from young mice to fill a lymphopenic environment. Therefore, differences in the thymic selection threshold in young versus adult mice skews the TCR repertoire, and the relatively higher self-reactivity of the T cell pool may contribute to the distinct immune responses observed in neonates.
Immunostimulation during primary neonatal RSV infection alters memory response.
Authors: Eichinger Katherine Marie , Kosanovich Jessica L , Manni Michelle L , Alcorn John F , Empey Kerry M
Published: January 2023

Respiratory syncytial virus (RSV) is recognized as a leading cause of acute viral illness in neonates worldwide. However, those with balanced Th1/Th2 responses, as a result of higher IFNγ concentrations, have milder disease and better clinical outcomes. The present study examined the effect of IFNγ administration during primary, neonatal RSV infection on innate and T cell responses during primary and secondary RSV exposure.

During primary infection, neonates treated with IFNγ had increased alveolar macrophage (AM) expression of CD80, MHCII, and scavenger receptor A. Subsequent CD8 T cell recruitment and activation was reduced in this group compared to RSV controls, however the RSV-specific CD8 T cell response was enhanced. Upon secondary infection, neutrophil and monocyte recruitment was boosted and expression of CD86 and MHCII was significantly increased on AMs with a correlative decline in CD73 expression compared to primary infection controls. In mice previously infected with RSV, T cell recruitment and activation was significantly increased. However, only those mice that were treated with IFNγ during primary infection showed improved CD8 T cell specificity following secondary RSV exposure. Viral lung titers were also significantly reduced in groups previously exposed to RSV but only IFNγ treatment during primary infection demonstrated significant improvements in airway resistance following methacholine challenge.

These results suggest that immunostimulation with IFNγ during primary neonatal RSV is associated with increased innate cell activation and improved CD8 T cell specificity. These cellular changes are associated with enhanced viral clearance and improved airway function.
A staphylococcal superantigen unexpectedly augments the response magnitude of cognate anti-influenza CD8+ T cells in mice and humans.
Authors: Meilleur Courtney Erin , Memarnejadian Arash , McCormick John , Haeryfar S.M. Mansour
Published: January 2023

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen (SAg) that induces proliferation and inflammatory cytokine secretion by up to 30% of T cells present in one’s repertoire. Eventually, the exposed T cells are thought to undergo activation-induced cell death or anergy, thus creating physical and/or functional holes in the repertoire. Using a mouse model of influenza A virus (IAV) vaccination, we found that primary IAV-specific CD8+ T cell (TCD8+) responses were enhanced, rather than diminished, in SEB-exposed animals. This effect was manifest for both local and systemic responses and validated when several different strains of IAV were utilized. Strikingly, the observed enhancement was easily detectable even one month post-SEB exposure, which was much later than the peak of the anti-IAV response. Importantly, IAV-specific TCD8+ in SEB-primed animals were not only capable of synthesizing interferon (IFN)-γ but also of lysing target cells pulsed with IAV-derived peptides in vivo. Finally, in vitro cultures of human peripheral blood mononuclear cells with SEB revealed that memory TCD8+, which recognize IAV but not Epstein-Barr virus or cytomegalovirus, were specifically expanded by this SAg and persisted long after deletion should have theoretically occurred. Taken together, our findings define a novel phenomenon in the context of TCD8+ responses to SAgs, which has important implications for vaccination against flu in critical care patients afflicted by staphylococcal infections and/or SAg-mediated illnesses.
Respiratory viral co-infections manipulate host immune responses to reduce disease severity.
Authors: Gonzalez Andres , Clary John , Rodriguez Anna , Miura Tanya
Published: January 2023

Patients with respiratory infections are frequently co-infected by more than one virus, but it is unknown how co-infection alters immune responses and disease outcomes compared to infection by the individual viruses. Our lab recently found that infection of mice with a mild rhinovirus pathogen (RV1B) two days before a more virulent virus (influenza A virus, PR8, or pneumonia virus of mice, PVM) reduced disease severity. In this study, we expanded these observations to determine how the timing of RV1B co-infection affects protection. Mice were inoculated with RV1B simultaneously with or two days after inoculation with PR8 or PVM. RV1B inoculated simultaneously with PR8 or PVM reduced disease whereas RV1B inoculated two days after PR8 and PVM increased the severity of PR8 and PVM. Next, mice were inoculated with RV1B two days before PR8 and then euthanized on days 4, 7 and 10 to quantify viral loads in the lungs. By day 7, co-infected mice had cleared PR8 while mice inoculated with PR8 alone had detectable virus. To identify immune cell populations that correspond with disease protection, groups of mice were inoculated with RV1B or mock two days before PR8 or PVM and flow cytometry analysis was performed on whole lungs on days 0, 2, 4, and 6 after the second virus. Co-infection by RV1B generated a stronger early innate response that contracted sooner than infection by PR8 or PVM alone. RV1B and PVM co-infection also had a stronger CD4 and CD8 T cell response later in infection. These data provide evidence that protection conferred by co-infection may be attributed to an earlier inflammatory cell response that contracts sooner, leading to faster clearance of the pathogenic virus while reducing immune-mediated pathology.
Influenza-specific ASC development is controlled by a T follicular helper cell dependent T-bet controlled feedforward loop.
Authors: Risley Chris A , Stone Sara L , Lund Frances E
Published: January 2023

Understanding the factors that drive differentiation of long-lived plasma cells (LLPCs) remains a significant roadblock en route to rationale vaccine design. Our lab previously described a prominent role for the cytokine-induced transcription factor, T-bet, in driving LLPC differentiation during a pulmonary viral infection. We observed that T-bet is upregulated by a significant proportion of virus-specific germinal center B cells (GCBs), which are the precursors of LLPCs. However, the cells and signals responsible for inducing T-bet and the T-bet transcriptional program in B cells remain unknown. It is likely that the interaction between the T follicular helper cells (TFHs) and the GCBs is primarily responsible for this programming. Drawing from research done with human samples, a subset of TFHs that express the T-bet regulated gene, CXCR3, has been described. We therefore hypothesize that these CXCR3+ TFHs express T-bet and are responsible for inducing T-bet expression by the GCBs and initiating T-bet transcriptional programming in the B cells. Studying the role of these CXCR3+ TFHs in mice gives us a unique opportunity to study the interaction between TFHs and GCBs, and determine how this specific interaction drives LLPC differentiation. Elucidation of these mechanisms will, in the future, allow us to address whether we can develop better and longer-lasting vaccines by choosing adjuvants that activate T-bet-dependent transcriptional programming in B cells.
Human parainfluenza virus type 3 encoded C protein antagonizes inflammasome activity
Authors: Shil Niraj Kanti , Pokharel Swechha Mainali , Thapa Bhim Bahadur , Bose Santanu
Published: January 2023

Human parainfluenza virus type 3 (HPIV3) is a negative-sense RNA virus in the paramyxovirus family and is the second leading viral cause of acute respiratory tract illness and hospitalization among infants and children worldwide. Interleukin-1β (IL-1β) is an important pro-inflammatory cytokine regulating innate immune response during infection. Inflammasome activation is required for IL-1β production. Inflammasome is a multi-protein complex platform that cleaves pro-caspase-1 to yield enzymatically active caspase-1. Caspase-1 in turn is involved in cleavage of pro-IL-1β into mature IL-1β. We recently showed activation of NLRP3/ASC inflammasome by HPIV3. NLRP3/ASC inflammasome activation triggered IL-1β production from HPIV3 infected macrophages. During our studies we identified HPIV3 encoded C protein as an inflammasome antagonist. C protein is an accessory protein originating from the over-lapping open reading frame of HPIV3 phosphoprotein (P) gene mRNA. Expression of C protein in human THP-1 macrophages led to diminished inflammasome activation and IL-1β production following treatment with LPS and nigericin. Similar result was observed following reconstitution of inflammasome in HEK293 cells expressing HPIV3 C protein. Mechanistically, our study revealed HPIV3 C protein regulating NLRP3 protein stability. Expression of C protein drastically reduced NLRP3 protein levels in THP-1 cells. Similar loss of NLRP3 protein was noted in HEK293 cells co-expressing C protein and NLRP3. However, C protein expression did not affect NLRP3 mRNA levels. Thus our studies have identified HPIV3 C protein as an inflammasome antagonist that directly modulates inflammasome activity by promoting NLRP3 protein degradation.
Host-immune responses to RSV infection and associated lung injury is modulated by Osteopontin (OPN) in age-dependent fashion
Authors: Durre Kainath , Qureshi Nagib , Qureshi Mahboob
Published: January 2023

RSV bronchiolitis and pneumonia causes significant mortality in young infants every year. The survivors become susceptible to asthma. In our present study, using a mouse model of BALB/C and NKT−/− mice, we examined the pulmonary responses to RSV infection in the very young hosts as opposed to relatively older hosts. We also examined the effects of exogenous osteopontin (OPN) administration on the alveolar cellular constitution by direct microscopy and flowcytometry and the role of NKT cells in this process. Pulmonary damage was assessed by measuring the Lactic Dehydrogenase (LDH) levels in the BALF by ELISA and histopathological examination by microscopy. Microscopy revealed a decreased lymphocyte response in the bronchoalevolar lavage fluid (BALF) of RSV-infected-4-day- old pups as compared to that of -14- and -20-day-old mice; but an enhanced neutrophil response, which was associated with increased LDH levels. Flowcytometry revealed that administration of OPN had downregulatory effects on lung lymphocyte (CD4+ and CD8+) responses in all age groups with proportional upregulation of lung macrophage (CD11bmed/hi, CD11Clo/−) responses. This effect was less pronounced in the older age groups. Additionally, activation of macrophages, as indicated by MHCII expression, was NKT dependent. Moreover, RSV- infection significantly inhibited lung CD8+ T cell activation as indicated by decreased CD44 expression, which was recovered by OPN administration. Understanding the underlying mechanisms of RSV-infection induced differential immune responses and OPN-mediated differential immunomodulation in different age groups may help develop newer therapeutic strategies.
IRF4-dependent DCs promote the establishment of CD8+ T cell memory during influenza infection.
Authors: AINSUA-ENRICH EROLA , Hatipoglu Ibrahim , Kadel Sapana , Turner Sean , Paul Jinny , Kovats Susan
Published: January 2023

The transcription factor IRF4 governs the development and function of CD11b+ dendritic cells (DCs). To determine the role of IRF4-dependent DCs in influenza infection, we compared responses of wild-type and CD11c-cre-Irf4f/f(KO) mice. The KO mice lack one lung-resident CD11b+ DC subset, and the other DC subsets lack IRF4. Upon infection with A/PuertoRico/8/34 virus, KO lung DCs contained increased IL-12 and reduced IL-10 RNA, which correlated with elevated numbers of CD103+ lung migratory DCs in the mLN. Consistent with this, KO mice showed greater numbers of T-bet+ and IFNg+ influenza antigen-specific CD8+ and CD4+ T cells, reduced numbers of Foxp3+ T regulatory cells, and decreased numbers of CD8+ T memory precursor cells. These altered effector T cell populations in KO mice impacted the memory T cell response to influenza virus. Numbers of virus-specific lung resident memory CD8+ T cells were significantly reduced in KO mice. Upon challenge with heterosubtypic virus, KO mice showed deficient expansion of virus-specific IFNγ+ CD8+ T cells. Taken together, these data show that KO mice mount a stronger antiviral T cell response upon primary infection but then have a weaker CD8+ T cell memory response to heterosubtypic virus. This supports the hypothesis that during influenza infection, the functional responses of IRF4-dependent DCs lead to increased differentiation of CD8+ memory precursor T cells and numbers of lung resident CD8+memory T cells.
Endogenous Secondary TCRs Promote Homeostatic Advantage Via Recognition of Self-Antigens
Authors: Balakrishnan Amritha , Jama Burhan , Morris Gerald P
Published: January 2023

Our laboratory studies how non-thymically selected secondary TCRs contribute to the T cell repertoire. We have previously demonstrated that peripheral T cells naturally expressing 2 TCRs have increased response to autoantigens. We hypothesized that this could promote homeostatic proliferation of T cells expressing 2 TCRs, which may cause autoimmune risk. To examine secondary TCRs in homeostasis, we performed competitive transfers of CFSE-labeled T cells from B6.Ly5.1 and B6.TCRα+/−.Thy1.1 (lacking secondary TCRs) into irradiated B6 recipients. T cells injected at a 1:1 ratio demonstrated a disadvantage for TCRα+/− T cells in lymphopenia-induced proliferation (LIP), with TCRα+/−/wt donor cell ratios of 0.6±0.1 at 8 d after transfer and 0.5±0.4 at 28 d after transfer. T cell population skewing resulted from increased proliferation of wild-type cells. The homeostatic advantage of secondary TCRs was less in chronic LIP (cLIP) (transfer into TCRα-knockout mice), with TCRα+/−/wt donor cell ratios of 0.8±0.3 at 8 d after transfer and 0.7±0.3 at 28 d after transfer. There was no difference between wt and TCRα+/− T cells in proliferative response to stimulation via anti-CD3/CD28, nor were differences observed for expression of CD3, CD5, or CD127. LIP and cLIP depend on signaling through the TCR, though reactivity to self-antigens is thought to drive LIP as opposed to endogenous antigens from commensal microflora in cLIP. Our results suggest that the advantage for secondary TCRs in homeostatic expansion reflects increased ability to interact with self-pMHC antigens. Current effort is focused to understand how this affects protective and autoreactive peripheral T cell repertoires during aging or iatrogenic lymphopenia.
The Intersection of Immune Responses, Microbiota and Pathogenesis in Giardiasis
Authors: Fink Marc Y , Maloney Jenny Grace , Keselman Aleksander M , Li Erqiu , Singer Steven M
Published: January 2023

Infection with the protozoan parasite, Giardia, can produce a range of symptoms including diarrhea, severe cramps, and nutrient malabsorption. Our lab and others have previously indicated that immune responses can contribute to pathogenesis. It has also been reported that intestinal microbiota can affect the ability of the parasite to colonize laboratory mice. Our goal was to determine if intestinal immune cells were activated following infection and how the microbiota interacted with intestinal immune responses.

Wild-type or CCR2 deficient mice were treated with a cocktail of antibiotics and infected with the GS strain. Control mice were not treated with antibiotics and/or were not infected. Changes in the intestinal microbiome and immune responses were determined using 16S sequencing and flow cytometry of isolated lamina propria and intraepithelial lymphocyte populations, respectively. Sucrase activity was also measured in mucosal lysates.

Mice treated with antibiotics or infected with Giardia exhibited shifts in their intestinal microbiomes. With or without antibiotics, we observed an increase in the frequency of CD4+ T cells in the lamina propria at day 7 post-infection. We also observed an increase in macrophage populations and activated CD8+ T cells, but only in mice not treated with antibiotics. Macrophage populations increased in wild-type and CCR2-deficient mice, and EdU labeling suggested in situ proliferation of resident macrophages. Finally, sucrase deficiency correlated with activation of CD8+ T cells.

Together these data indicate that the intestinal microbiota contribute to immune cell activation during infections and suggest that the microbiota may contribute to different infection outcomes.
Progression of experimental autoimmune encephalomyelitis (EAE) increases manganese superoxide dismutase (MnSOD) protein in the mitochondria of the mouse renal cortex
Authors: Zhou Xiaoming , Packialakshmi Balamurugan
Published: January 2023

Sodium has been implicated in the high NaCl diet-induced exacerbation of EAE. The kidney cortex is the primary site for regulating Na homeostasis. Reabsorption of Na by the cortex absolutely relies on the ATP-dependent Na,K-ATPase. Our previous studies demonstrated that the progression of EAE increases the Na,K-ATPase activity in the cortex even under normal salt intake. Mitochondrial respiration not only generates ATP, but also produces reactive oxygen species (ROS). As MnSOD is the first line of defense against the harmful effect of mitochondrial ROS, the present study analyzed whether EAE had any effects on MnSOD. Progression of EAE from mild to severe stage increased total MnSOD protein by 38% and had no significant effect on CuZnSOD protein. This effect was not explainable solely by the illness-induced reduction of water or food intake. Further analyses reveals that severe EAE increased mitochondrial MnSOD protein by 47%, Na,K-ATPase beta1 subunit protein by 64% with a concomitant increase of mitochondrial function as measured by the complex II activity and protein level of cytochrome oxidase 4, a major component of complex IV. Activation of Na,K-ATPase with two mechanistically different reagents, monensin and dexamethasone, simulates the effect of EAE on MnSOD protein in HEK293 cells. Fluorescent microscopic study showed that monensin and dexamethasone increased ROS in HEK293 cells, and this effect was inhibited by 400 U/ml catalase. Similarly, catalase also erased the effects of monensin and dexamethasone on MnSOD protein. In conclusion, progression of EAE increases mitochondrial MnSOD protein in the kidney cortex, likely due to the ROS produced during respiration to meet the demands of ATP for the Na,K-ATPase.
Selective inhibition of NF-kB inducing kinase (NIK) is therapeutically efficacious in IFNα-accelerated lupus nephritis prone mice
Authors: Brightbill Hans D , Suto Eric , Blaquiere Nicole , Ramamoorthi Nandhini , Sujatha-Bhaskar Swathi , Gogol Emily , Castanedo Georgette , Jackson Benjamin , Haller Susan , Lesch Justin , Bents Karin , Everett Christine , Kohli Pawan Bir , Linge Sandra , Christian Laura , Barrett Kathy , Jaochico Allan , Berezhkovskiy Leonid M , Fan Peter W , Veliz Kelli , Townsend Michael , DeVoss Jason , Johnson Adam , Godemann Robert , Lee Wyne P , Austin Cary D , McKenzie Brent , Hackney Jason A , Crawford James J , Staben Steven , Ismaili Moulay Hicham Alaoui , Wu Lawren C , Ghilardi Nico
Published: January 2023

Systemic lupus erythematosus (SLE) is often considered a disease driven by autoreactive B cells and anti-nuclear antibodies. However, therapeutic targeting of B cells, for example through BAFF blockade, has been only partially effective in SLE. NF-kB Inducing Kinase (NIK) mediates non-canonical NF-kB signaling downstream of disease relevant TNF family members, such as BAFF, TWEAK, CD40, and OX40. We hypothesized that NIK inhibition might be more efficacious than BAFF blockade in lupus, and therefore generated a highly selective and potent small molecule inhibitor (SMI) of NIK to test this hypothesis. In cellular assays, this molecule inhibits non-canonical NF-kB signaling downstream of multiple TNFRSF family members. In order to differentiate the effects of NIK inhibition and BAFF blockade in vivo, we compared NIK inhibition with BAFF blockade in the context of NZB/W F1 lupus prone mice. As predicted, NIK inhibition recapitulated the pharmacological effects of BAFF blockade. Furthermore, NIK inhibition, but not BAFF blockade, affected T cell parameters in the spleen and pro-inflammatory gene expression in the kidney, the latter of which is partly attributable to TWEAK signaling. Finally, NIK inhibition resulted in improved survival, ameliorated renal pathology, and lower proteinuria scores. Collectively, our data suggest that NIK inhibition affects multiple disease-relevant pathways and may therefore have superior efficacy compared to BAFF inhibition in SLE.
Prostaglandin E2 receptor (EP2): a novel target to attenuate excessive bone loss during autoimmune arthritis
Authors: Ignatowicz Aleksandra P , Patterson Daryllynn C , Mula Ramanjaneya V R , Dingledine Raymond J , Ganesh Thota , Shashidharamurthy Rangaiah
Published: January 2023

Prostaglandin-E2 receptors (sub type EP2) are known to be activated during various autoimmune inflammatory disorders including rheumatoid arthritis (RA) and may play an essential role in exacerbating the bone damage during RA. Recently, we have reported that EP2 antagonists inhibited pro-inflammatory cytokine responses at the transcriptional level using murine monocytic cell line and observed that EP2 antagonists attenuate the upregulation of several inflammatory mediators in vitro. It has been shown that EP2 gene deletion in mouse cultures impairs osteoclast formation. Herein we investigated the anti-osteoclastogenic activity of recently discovered EP2 antagonists using an in vitro osteoclastogenesis model using mouse monocytic cell line (RAW264.7 cells). We observed significantly increased size and number of osteoclasts by both PGE2 and butaprost (selective EP2 agonist) compared to receptor activator of nuclear factor kappa-B ligand (RANKL) alone treated cells. We did not observe significant difference in number of osteoclasts between PGE2 and butaprost. In addition, 10μM concentration of various EP2 specific antagonists (TG8-4, TG4-155 and TG6-129) inhibited RANKL-induced osteoclast formation. Western blot analysis revealed that EP2 antagonists decreased the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are the master regulators of osteoclastogenesis. These data indicates the direct effect of EP2 antagonists on bone cells in preventing the severe bone damage implying EP2 receptors play a major role during osteoclast formation, thus EP receptors should be explored as a therapeutic target to blunt the excessive bone loss during arthritis.
Transcriptome analysis reveals persistent effects of neonatal diet on the ileum gene expression in porcine neonates
Authors: Saraf Manish Kumar , Chen Celin , Bowlin Anne Kidd , Dawson Harry , Badger Thomas M , Yeruva V. Laxmi
Published: January 2023

Mechanism(s) underlying long-lasting effects of human breast milk (HBM) on gut development and immune function have not been investigated. To differentiate between HBM and formula diet’s persistent effect on gene expression of a small intestine, piglets (n=15/group) were fed either HBM or dairy-based formula (FM) from postnatal day 2 to 21, followed by a soy free pig wean diet until day 51. Relative to HBM-fed, FM-fed had heavier small intestine (436±15.3 vs 362±45 g, p<0.05) but no change in the length. Transcriptome analysis revealed that the identified genes associated with cytokines (4↑, 9↓; i.e. CXCL8↓), Wnt (2↑, 9↓; CDHR2↓), gonadotropin releasing hormone receptor (1↑, 10↓; PRLR↓), EGF receptor (1↑, 7↓; CBL↓), cadherin (7↓; FRK↓) and endothelins (7↓; PRKG2↓) signaling were differentially expressed (2–5 fold) in ileum (IL) of FM-fed relative to HBM-fed. Moreover, the genes involved in biological processes such as cellular (84↑, 138↓; 2–12 fold), metabolic (71↑, 104↓; 2–17 fold), immune (14↑, 27↓; 2–10 fold) and developmental (16↑, 40↓ genes; 2–6.8 fold) were differentially expressed in IL of FM-fed relative to HBM-fed. Genes associated with T- (BRAF) and B-cell (BRAF, ITPR3 and FRK) activation were also decreased in IL of FM-fed relative to HBM-fed. NK, B and T-reg cells populations were reduced in mesenteric lymph nodes of FM-fed relative to HBM-fed. In summary, the data suggest a persistent effect of FM and HBM diets on small intestine and diet-driven changes in specific pathways of a small intestine. HBM may regulate these pathways to favor the gut development and immune function. However, FM may not harmonize it well and thereby delay the physiological process. Microbiome role is being evaluated to understand a specific mechanism.
Gut microbiota as a source of signals that trigger spontaneous ocular autoimmunity.
Authors: Salvador Ryan S. , Horai Reiko , Tang Jihong , Zárate-Bladés Carlos , Jittayasothorn Yingyos , Itoh Kikuji , Umesaki Yoshinori , Caspi Rachel R
Published: January 2023

Autoimmune uveitis, a major cause of blindness, is thought to be driven by T cells specific for unique retinal antigens that have been activated and acquired the ability to cross the blood-retinal barrier. However, it is unknown where and how they first become activated. The R161H mouse expresses a transgenic TCR specific for the retinal protein IRBP and develops spontaneous uveitis with 100% incidence, permitting to study natural triggers of disease. Retina-specific T cells were activated in the intestine of specific-pathogen-free (SPF) R161H mice. Elimination of gut commensals by oral treatment with antibiotics (ABX) or by rearing under germ-free conditions (GF) significantly attenuated uveitis and reduced Th17 cells in the gut lamina propria. Bacteria-rich extracts of intestinal contents from SPF, but not GF or ABX mice, activated retina-specific T cells in vitro, and R161H T cells signaled through the clonotypic TCR in the gut in vivo, suggesting a role for gut microbiota as a source of stimulating signals for retina-specific T cells. To dissect the contribution of adaptive vs innate microbial signals, gnotobiotic studies were performed: GF R161H mice exposed to SPF conditions developed full-blown uveitis, but mono-colonization with segmented filamentous bacteria (SFB) or a Turicibacter strain (T. H121) only partially restored disease. Unlike intestinal extracts from SPF mice, extracts from monocolonized mice failed to activate R161H T cells in vitro, suggesting lack of “antigen” activity, but SFB (not T. H121) restored gut Th17-producing cells, indicating presence of “adjuvant” activity. Thus, microbial-derived adaptive “antigen” and innate “adjuvant” signals are both required for full development of uveitis.
Paneth Cell Specific Lysozyme Regulates Intestinal Mucosal Immune Response by Shaping Gut Microbiota Landscape
Authors: Yu Shiyan , Douard Veronique , Edelblum Karen L. , Laubitz Daniel , Zhao Yanlin , Kiela Pawel R , Yap George S , Gao Nan
Published: January 2023

Paneth cells produce antimicrobial proteins such as lysozyme and cytokines to interact with gut microbiota and mucosal immune system respectively, maintaining delicate mucosal immune homeostasis. Abnormalities in lysozyme expression or intracellular distribution have been cataloged in patients with inflammatory bowel disease (IBD) and mouse IBD models. However, it is unclear whether these defects are simply the consequences of IBD pathogenesis or they may also contribute to disease progression. To explore the role of Paneth cell lysozyme in such a microbe-friendly environment, we derived two mouse models: Paneth cell Lyz1 knockout (Lyz1KO) and gut epithelial Lyz1 overexpression (Villin-Lyz1). Using these two models, we performed fecal 16s rRNA sequencing and found that the abundance of several mucolytic bacteria (e.g. D. formicigenerans and R. gnavus) was increased in Lyz1KO mice but decreased in Villin-Lyz1, as confirmed by species specific qPCR. In addition, we confirmed that lysozyme could inhibit the growth of D. formicigenerans and R. gnavus in vitro. Interestingly, the number of goblet and tuft cell in Lyz1KO mice was increased compared to that in wildtype littermates. In line with this, type 2 cytokines (e.g. Il25 and Il13) were elevated in Lyz1KO mice. Antibiotics cocktail treatment rescued goblet and tuft cell hyperplasia, suggesting altered gut microbiota landscape is a critical factor inducing goblet and tuft cell hyperplasia. Consistently, disruption of intestinal type 2 immunity axis by anti-CD90.2 or anti-Il13 neutralizing antibody also ameliorated goblet and tuft cell hyperplasia. Taken together, our findings suggest lysozyme modulates intestinal type 2 immune response via shaping gut microbiota landscape.
Microbial and Maternal Factors Control the Development of RORγt+ Regulatory T Cells Promoting Durable Tolerance and Preventing Allergy
Authors: Knoop Kathryn A , McDonald Keely , Gustafsson Jenny , Hogan Simon P , Elson Charles O , Tarr Philip I , Newberry Rodney D
Published: January 2023

Mounting evidence emphasizes the importance of exposure to food allergens and gut microbes in early life to promote tolerance and reduce the risk of food allergy. Indeed the NIAID recently recommended early introduction of dietary peanut to reduce peanut allergy in at risk children. The mechanisms promoting tolerance during early life are incompletely understood, but potentially relate to a population of long-lived RORγt+ inducible regulatory T cells (iTregs) which prevent the development of food-specific Th2 responses throughout life. The optimal time for exposure to environmental antigens for the development of these RORγt+ iTregs, where in the gut protective exposures occur, and how the immune system’s contact with these antigens is controlled in early life remain unknown. We identified a window of time in early life in which luminal antigens are shunted to the colonic lamina propria (LP) and delivered to colonic LP antigen presenting cells by goblet cell-associated antigen passages (GAPs), correlating to an expansion of colonic LP RORγt+ iTregs. GAP formation was regulated by goblet cell sensing of breast milk epidermal growth factor and the blooming gut microbiota. Disruption of the gut microbiota or GAP formation and antigen delivery during early life abrogated tolerance to dietary antigens, decreased RORγt+ iTregs, and induced a skewing of the immune system toward systemic Th2 (allergic) responses. We propose the gut microbiota, in concert with maternal factors, control antigen delivery and the induction of RORγt+ iTregs during infancy. These mechanisms allow for proper education of the immune system against reaction to dietary and microbial antigens, a process that then suppresses inappropriate Th2 responses later in life.
GPR43 mediates microbiota metabolite SCFA induction of antimicrobial peptide expression in intestinal epithelial cells via activation of mTOR and STAT3
Authors: Chen Feidi , Zhao Ye , Huang Xiangsheng , Chen Liang , Sun Mingming , Yao Suxia , Xiao Yi , Cong Yingzi
Published: January 2023

As a critical component in maintaining intestinal homeostasis, intestinal epithelial cells (IEC) are known to tune gut microbiota via antimicrobial peptides (AMP) production. In return, microbiota has been reported to regulate IEC expression of AMPs, including RegIIIg and certain defensins. The underlying mechanisms are, however, still not completely understood. The present study attempted to address the novel pathways by which gut microbiota regulates IEC expression of AMP as a way to contribute to intestinal homeostasis. We found that the mice with deficiency of GPR43, a receptor for short chain fatty acids (SCFA), the metabolites of gut microbiota, have lower expression of RegIIIg and b-defensins 1, 3, and 4 in IECs compared to that of WT mice. Oral feeding with SCFA promoted IEC production of both RegIIIg and defensins in mice. Moreover, ex-vivo culture of enteroid revealed that SCFA induction of IEC RegIIIg and b-defensins production is GPR43-dependent. Mechanistically, SCFA activated mTOR and STAT3 in IECs. Knockdown of either mTOR or STAT3 impaired SCFA-induced AMP production. Collectively, our data revealed that microbiota-derived SCFA promote IEC production of both RegIIIg and b-defensins, which is through GPR43 and mediated by mTOR and STAT3 pathways.
Vitamin A deficiency impacts acquisition, but not expression, of autoimmunity to the neuroretina
Authors: Horai Reiko , Zhou Ru , Bing So Jin , Wloka Kaska , Chen Jun , Silver Phyllis , Jittayasothorn Yingyos , Caspi Rachel R
Published: January 2023

Vitamin A (VitA) and its derivative retinoic acid (RA) are essential for immunological responses. Acquisition of effector responses is impeded in VitA-deficient (VAD) mice, but little is known about maintenance and expression of previously acquired effector function in the VAD environment, or its impact on progression of autoimmune diseases. We examined this using two models of uveitis: experimental autoimmune uveitis (EAU) induced by active immunization and spontaneous uveitis in retina-specific T cell receptor transgenic (R161H) mice, and in the model of experimental autoimmune encephalomyelitis (EAE). VAD was induced by dietary lack of VitA from before birth, or by daily injections of a pan-RA receptor inhibitor BMS493 in adult mice fed with standard diet. VAD mice were essentially resistant to induction of EAU or EAE and displayed impaired effector T cell responses. Defective priming/acquisition of effector function by VAD T cells was also evident in vitro. Interestingly, however, effector T cells primed in a VitA-sufficient environment were able to function in VAD recipients and induced EAU. Furthermore, spontaneously uveitic R161H mice fed with VAD diet, in which priming of pathogenic T cells occurs before onset of full VitA deficiency, appeared to develop unreduced or even exacerbated spontaneous uveitis compared to VitA-sufficient R161H mice. We conclude that although priming of naïve T cells in the VAD environment is defective, effector function acquired under VitA-sufficient conditions is maintained and can be expressed under VAD conditions. Because dietary lack of VitA is rarely profound and may be seasonal, our findings may shed light on immunity and autoimmunity in geographical regions where dietary VitA is limiting.
A serum CNS-specific protein fingerprint predictive of clinical relapses and disease development of multiple sclerosis
Authors: Raphael Itay , Gomez-Rivera Francisco , Huizar Carol Chase , Gupta Rishein , Arulanandam Bernard , Forsthuber Thomas
Published: January 2023

There is a critical need in MS to develop biomarkers to improve early diagnosis, predict imminent disease relapses, and optimize treatment responses. In MS, central nervous system (CNS) inflammation results in damage to neuronal tissues and disruption of blood-brain barrier integrity, and ultimately to leakage of CNS-specific proteins (CSPs) into serum. Therefore, altered serum levels of CSPs could be promising biomarkers of MS. Based on our previous discovery of CSP expression waves in brain tissue of mice with experimental autoimmune encephalomyelitis (EAE), a preclinical model for human MS, we investigated a subset of CSPs which have human-homologs in longitudinal serum specimens from individual mice in this model. Herein, we establish a pre-onset serum CSP expression wave that enabled prediction of clinical onset of EAE and stratification of subjects into diseased versus healthy. Importantly, we demonstrate that the pre-onset serum CSP wave was not affected by adjuvants and microbial infection, thereby has high sensitivity and specificity for a neuroinflammatory disease. Moreover, we identify differences in serum protein expression between active and passive immunization of EAE revealing hitherto not appreciated differences for disease induction mechanisms. The results provide proof of concept for the development serum CSP waves as biomarkers of MS, specifically for early detection and prediction of the onset of clinical relapse.
A Role for the CD27-CD70 Pathway in T Cell Driven Skin Inflammation
Authors: Remedios Kelly Anne , Zirak Bahar , Rosenblum Michael D
Published: January 2023

The CD27-CD70 costimulatory pathway plays important roles in T cell function and has been implicated in multiple autoimmune diseases. It is currently unknown whether this costimulatory pathway influences T cell responses in the skin. To study the role of CD27 in CD4 T cells, we have utilized the previously described K5 TGO model of inducible tissue-antigen expression. In this model, mice with both K5 and TGO transgenes allow the expression of the model antigen ovalbumin (ova) to be stringently induced or extinguished in the epidermis through the application or withdrawal of doxycycline. After turning on antigen, the transfer of WT ova-specific CD4 (OTII) T cells into K5/TGO recipients induces an inflammatory dermatitis characterized by scaling and neutrophil influx. When CD27-deficient OTII T cells were transferred, skin disease was attenuated and neutrophil influx was decreased. CD27 costimulation promoted T cell accumulation in the skin draining lymph nodes and skin and IL-2 and IFNg production upon restimulation ex vivo. Using in vivo competition experiments, we found that CD27 promoted CD4 responses in a cell-intrinsic manner. CD27 had minimal effect on early T cell activation and proliferation but seemed instead to be involved in promoting survival. Our studies highlight a previously un-identified role for the CD27 pathway in promoting CD4 T cell responses in the skin.
PKC-θ is dispensable for OX40L-induced TCR-independent Treg proliferation
Authors: Alharshawi Khaled , Marinelarena Alejandra , Kumar Prabhakaran , El-Sayed Osama , Bhattacharya Palash , Sun Zuoming , Epstein Alan L. , Prabhakar Bellur S.
Published: January 2023

Previously, we have shown that GM-CSF-induced Bone marrow-derived dendritic cells (G-BMDCs), upon co-culture with splenic CD4+ T cells, have the capability to induce preferential proliferation of regulatory T cells (Tregs) in TCR-independent and OX40L/OX40 dependent manner. In this study we investigated the signaling involved in this Treg proliferation induced by G-BMDCs. Firstly, using splenic CD4+ T cells from OX40 deficient mice and G-BMDCs from WT mice, we confirmed the important role of OX40L/OX40 interaction in Treg expansion. Since OX40 stimulation in the absence of TCR engagement has been reported to induce the assembly of a signalosome involving PKC-θ, we investigated the requirement of PKC-θ in our Treg proliferation mechanism. Interestingly, PKC-θ deficient-CD4+ T cells showed impaired Treg proliferation upon co-culture with WT G-BMDCs. However, this defect in proliferation could be rescued upon IL-2 addition suggesting that PKC-θ was dispensable for Treg proliferation. We further demonstrated that PKC-θ was essential for T-effectors to provide IL-2, which was required for OX40L/OX40-induced Treg proliferation. Furthermore, injecting WT, OX40- deficient, and PKC-θ-deficient mice with soluble OX40L, we established that OX40L/OX40 signaling was sufficient to induce in vivo expansion of Tregs in WT and PKC- deficient mice, but not in OX40-deficient mice. Finally, our data suggested a possible involvement of TRAF1 in Tregs, downstream of OX40 activation.