bluuebunny/crossref_metadata_2025 huggingface dataset:
Maternal EGF protects offspring from enterally acquired bloodstream infections by limiting bacterial translocation in the neonatal intestine
Authors: Knoop Kathryn A , Floyd Alex , Warner Barbara , Tarr Phillip , Newberry Rodney D
Published: January 2023

Late-onset neonatal sepsis (LOS), a significant cause of morbidity and mortality in prematurely born infants, results from a bloodstream infection of gut-originating bacteria. It remains unclear how such bacteria translocate from the intestine, though it has been repeatedly observed that breastfed infants, particularly those fed mother’s own milk (MOM), have reduced risk of LOS compared to formula fed infants. Epidermal growth factor (EGF) promotes intestinal barrier function in infants and is present at high concentrations in breast milk post-partum and decreases throughout lactation. We found reduced concentrations of EGF in the stool of premature formula-fed infants compared to MOM-fed infants, and observed a similar decrease in EGF concentrations of stool of neonatal mice asynchronously cross-fostered (ACF) to dams that had delivered two weeks prior. LOS bloodstream isolates of E. coli colonized the tracts of all pups but translocated and disseminated systemically only in ACF mice resulting in bacteremia and rapid death. Goblet cell intrinsic sensing of EGF via the epidermal growth factor receptor (EGFR) limited bacterial translocation from the colon. Oral gavage of recombinant EGF reduced bacteria translocation and prevented the development of systemic disease in ACF mice. Thus, disruption of maternally delivered EGF in ACF mice results in translocation of pathogens from the gut, and a sepsis-like disease. In conclusion we have identified a mechanism whereby a gut-residing pathogen gains systemic access and have developed a novel animal model replicating this mechanism to explore the protective effect of breastmilk and EGF in LOS.
Diverse dendritic cell subsets promote infection and induction of CD8 T cell responses after foodborne Listeria monocytogenes infection
Authors: Imperato Jessica Nancy , Xu Daqi , Romagnoli Pablo A , Qiu Zhijuan , Perez Pedro , Khairallah Camille F , Pham Quynh-Mai , Milling Simon W.F. , Lefrancois Leo , Khanna Kamal M , Puddington Lynn , Sheridan Brian S
Published: January 2023

Listeria monocytogenes (Lm) is a common foodborne pathogen which causes mild gastrointestinal distress in healthy hosts, and systemic listeriosis in immunocompromised individuals. While immune responses have been rigorously examined after intravenous Lm infection, less is understood about Lm dissemination from the intestines or the induction of adaptive immune responses after physiologic infection through the consumption of contaminated food. Consequently, this study focused on the cells involved in early immune responses in the intestinal mucosa using foodborne Lm infection in mice. After foodborne infection, Lm trafficked intracellularly from the intestines to the mesenteric lymph nodes (MLN) and were associated with Batf3-independent dendritic cells (DC) in the lymphatics. Consistent with this observation, dissemination of Lm from the gut to the MLN occurred normally in Batf3−/− mice. Activated migratory DC accumulated in the MLN by 3 days post-infection (dpi) where they surrounded Lm foci. At this time, Lm burden in the MLN was reduced in Batf3−/− mice implicating Batf3-DC in the maximal accumulation of Lm. While both conventional type 1 DC (cDC1) and type 2 DC in the MLN contained Lm by 3 dpi, Batf3−/− mice exhibited a profound defect in the induction and gut homing of Lm-specific CD8 T cells. Surprisingly, restoration of pathogen burden was unable to restore CD8 T cell responses in Batf3−/− mice. Thus, cDC1 play a critical role in the generation of potent anti-Lm immunity following foodborne infection. Collectively, these data demonstrate that DC play diverse and dynamic roles in the early events that follow foodborne Lm infection, and in the establishment of protective, anti-Lm intestinal immunity.
The inflammatory response after an intestinal inflammatory challenge affects in an opposite manner to the female and male offspring gestated in hypothyroxinemia
Authors: Rivera Juan Carlos , Gutiérrez Barbara , Díaz Máximo , Chamorro Linda , Estrella Matías , Guajardo Reynaldo , Díaz Ignacio , Opazo María Cecilia , Boudin Helene , Neunlist Michel , Bueno Susan M , González Pablo , Kalergis Alexis M , Riedel Claudia
Published: January 2023

Ulcerative colitis (UC) is a highly frequent inflammatory bowel disease (IBD), which causes are unknown. We have shown that the offspring gestated under thyroxine (T4) deficiency, condition named hypothyroxinemia (HTX), shows inflammatory features at the intestine. Moreover, the offspring gestated under HTX (HTX-offspring) suffers strong experimental autoimmune disease. Suggesting that gestational HTX could imprint their offspring with a high immune response after an inflammatory challenge. Thus, we think that the HTX-offspring will be more prone to suffer IBD. To analyze this hypothesis C57BL6 pregnant mice were induced with gestational HTX during E10 to E15 by giving them methimazole in the tap-drinking water. Then, UC was induced to the HTX-offspring or control at postnatal P55 by giving them dextran sodium sulphate (DSS) in the tap-drinking water for six days. Body weight, pathological score of IBD and hidden blood in stool were analyzed daily. Then, mice were sacrificed at day 10 to evaluate their inflammatory response. For that histological analysis were performed in the intestine; the content of cytokines in serum and intestine by ELISA and the populations of innate and adaptive immune cells at the intestine and spleen by flow cytometry. Our results shown that the male and female offspring developed an opposite immune response to UC. The female HTX-offspring were more resistant and the male HTX-offspring were more sensitive to UC compared to their respective female and male control groups. Thus, our results suggest that the immune response of an individual can be set up during gestation and that the normal level of maternal T4 during gestation is fundamental for the proper function of the offspring immune system.
Toll-like receptor 7 protects against intestinal inflammation and restricts the development of tissue-resident memory CD8+ T cells
Authors: Hamade Hussein , Stamps Jasmine , Thomas Lisa , Stamps Dalton , Tsuda Masato , Oshima Naoki , Wong Michelle , Salumbides Brenda , Jin Caroline , Nunnelee Jordan , Targan Stephan R , Michelsen Kathrin S
Published: January 2023

The maintenance of intestinal homeostasis depends on a complex interaction between microbiota, intestinal epithelial barrier, and the immune system. Alteration in of one of these components could lead to the development of inflammatory bowel diseases (IBD). Norovirus infection of mice with a mutation in the Crohn’s disease susceptibility gene Atg16L1 induces intestinal inflammation. Moreover, persistent norovirus infection leads to intestinal virus-specific CD8+ T cells responses. However, the role of the enteric virome in IBD is still poorly understood. Toll-like receptor 7 (TLR7) recognizes single-stranded RNA viruses. Here, we investigate the role of TLR7 in intestinal homeostasis and inflammation. At steady state, Tlr7−/− mice have an approx. 10-fold increase in small and large intestinal lamina propria (LP) granzyme B+ tissue-resident memory (Trm) CD8+ T cells compared to WT mice (WT: 5.5, Tlr7−/−: 59.5 %, p < 0.005), reminiscent of persistent norovirus infection. Furthermore, Tlr7−/− mice were more susceptible to dextran sulfate sodium (DSS) colitis with more severe inflammation (Histoscore: WT: 7.6, Tlr7−/−: 12.7, p < 0.005), increased disease activity index (WT: 5.5, Tlr7−/−: 7.4, p < 0.05), and increased secretion of IFNg (WT: 5.2, Tlr7−/−: 24.2 ng/ml, p < 0.005) and TNFα (WT: 108.6, Tlr7−/−: 191.8 pg/ml, p < 0.05). Increased colonic inflammation was associated with increased LP Trm CD8+ T cells (WT: 3.9, Tlr7−/−: 42.0 %, p < 0.005). Our data shows that TLR7-deficiency promotes the development of LP Trm CD8+ T cells and increases susceptibility to DSS colitis. In conclusion, TLR7 plays an important role in maintaining immune response to intestinal viruses and protects against development of colitis.
Integrin β7 is required for B cell migration and regulation of the intestinal microbiota during colitis
Authors: Tyler Christopher J , Guzman Mauricio , Lundborg Luke , Yeasmin Shaila , Rivera-Nieves Jesus
Published: January 2023

The incidence of ulcerative colitis (UC) is increasing worldwide. A proven therapeutic strategy for UC is the inhibition of leukocyte trafficking via blockade of integrin β7. Despite this, the mechanism of action of vedolizumab (anti-α4β7) is poorly defined. Clear definitions of the immune subsets targeted by vedolizumab are essential for understanding the pathogenesis of UC, and development of biological endpoints for assessing treatment efficacy.

Methods

The IL-10−/− colitis model was crossed with integrin β7 deficient mice, and clinical features and histological scores were recorded. Flow cytometry of B-cell populations was conducted in colonic cell isolations. 16S bacterial sequencing was conducted on intestinal content.

Results

IL-10−/−β7−/− mice displayed increased mortality compared to IL-10−/− mice at 12 weeks and uniformly developed rectal prolapses. IL-10−/−β7−/− mice displayed increased colonic inflammatory indices (n>10, p<0.001), associated with a reduced B-cell, but normal T-cell, intestinal infiltrate (n>6, p<0.001). IL-10−/−β7−/− mice displayed a resulting IgA deficiency and significant alterations to the resident microbiota. The presence of systemic bacterial infection was observed. Blockade of the α4β7 ligand MAdCAM-1 in IL-10−/− mice resulted in a parallel phenotype.

Discussion

Integrin β7 deficiency results in a worsening of colitis in mice, resulting from a B-cell deficit in the inflamed colon and control the microbiota, via IgA. B-cells are largely ignored in the field of IBD and the potential complications associated with chronic integrin blockade. Our data suggests that the role of integrin β7 on B-cell migration may have important implications on the pathogenesis of colitis in mice and humans.
Antagonists of chemokine receptor CCR9 synergize with anti-TNFα immunotherapy to reduce inflammation in the MDR1a−/− mouse model of colitis
Authors: Campbell James J , Ertl Linda S , Ebsworth Karen J , McMahon Jeffrey P , Zhang Penglie J , Chhina Vicky J , Charo Israel F , Schall Thomas J
Published: January 2023

The development of anti-TNFα immunotherapies has led to great advances in quality of life for Crohn’s and colitis patients, and has allowed a reduction in steroid use for these diseases. However, a significant proportion of IBD patients is resistant to anti-TNFα agents, or becomes resistant over time. Preclinical research and clinical trials suggest that CCR9 antagonism can effectively reduce colon inflammation. CCR9 is expressed on a subset of T cells dedicated to intestinal trafficking. Antagonism of this receptor inhibits the accumulation of T cells within inflamed intestinal epithelium and lamina propria. Since anti-TNFα and CCR9 antagonists act through entirely different mechanisms, we asked whether these two approaches might synergize and expand the effectiveness and scope of each therapy. We used a piroxicam-accelerated version of the MDR1a−/− spontaneous colitis model in mice. We have found that combined dosing of piroxicam-fed MDR1a−/− mice with a CCR9 antagonist and an anti-TNFα MAb significantly reverses loss of body weight, diarrhea score and colon inflammation. The combined dosing was more effective than anti-TNFα or CCR9 antagonist alone, as measured by colon length/weight ratio, neutrophil accumulation in the colonic epithelium and reversal of the loss in body weight characteristic of this model. These data suggest that combining a CCR9 antagonist with existing anti-TNFα treatment regimens may extend the benefits of anti-TNFα immunotherapy to a larger class of patients suffering from IBD, and may prolong the effectiveness of anti-TNFα immunotherapy for those patients already undergoing such treatments.
Exosomal miRNA19a from primary human nasal epithelial cells induce pro-inflammatory responses of macrophages via RORA
Authors: Shin Cheolhee , Tran Na Ly , Kim Tae Hoon , Kim Sang-Heon , Oh Seung Ja
Published: January 2023

Macrophages are phagocytes that play a critical role in the immune system. They are involved in both innate and adaptive immunity by either removing pathogens or antigen presentation. It has been shown that macrophages are polarized to subgroups with distinct functions, M1 and M2. M1 polarization is characterized by pro-inflammatory and anti-cancer functions, whereas M2 macrophages promote immune suppression and tumor growth. In this study, we investigate that exosomal miRNAs from primary human nasal epithelial cells play roles in airway mucosal microenvironment. Specifically, we demonstrated that the human nasal epithelial cells treated with urban dust modulate airway mucosal microenvironment through upregulation and release of miRNAs. The result shows that the human nasal epithelial cells treated with urban dust altered miRNA expression profile and released miRNAs promotes M1 macrophage polarization. The study will further investigate exosomal miRNA mediated-communications between epithelium and macrophages in airway mucosal microenvironment.
Klebsiella Pneumoniae Mucosal Vaccination Elicits Lung CD4+ TRM cells that are resistant to CD4 depleting antibodies
Authors: Iwanaga Naoki , Sandquist Ivy , Chen Kong , Norton Elizabeth B , Rangel-Moreno Javier , Kolls Jay K.
Published: January 2023

Objective

Mucosal immunization with a Klebsiella pneumoniae (Kp) vaccine elicits noble CXCR6+CD4+ T cells that mediate serotype independent immunity, but the stability of this response is unclear. Thus we conducted experiments to determine if these cells circulate and whether they could be depleted by a depleting anti-GK1.5 mAb.

Methods

Mice were vaccinated via intratracheal with Kp serotype 2 OMPx adjuvanted with heat-labile enterotoxin A1 domain (LTA1) and boosted at 3 weeks after the initial immunization. T-cell responses were evaluated by FACS and ELISPOT. CD4+ T cell depletion in the immunized lung, lymph nodes, and spleen by administering 300ug anti-GK1.5 mAb intraperitoneally was examined using a secondary antibody for anti-GK1.5 mAb. Finally, lung and spleen CD4+ T cells of intratracheally immunized CD45.1+ C57BL/6 mice were transferred to CD45.2+ C57BL/6 mice and Rag2−/− mice.

Results

Immunized lung CD4+ T cells were increased equivalently in both C57BL/6 mice and Cxcr6−/− mice, demonstrating that CXCR6 was dispensable for the formation of lung CD4+ T cells. Splenic CD4+ T cells were > 95% depleted with anti-GK1.5 mAb. In contrast, < 5% of vaccine elicited lung CD4+ T cells were depleted despite being well coated by the antibody, strongly suggesting that these cells are TRM cells. Additionally, using an adoptive transfer model, we found that lung CD4+ T cells preferentially homed to lung compared to splenic CD4+ T cells. We next performed single-cell RNAseq on these cells and elucidated several unique adhesion molecules such as Embigin, Ramp1, Lgals1, and Thy1 besides Cxcr6.

Conclusions

These data support that vaccine strategies can be developed by targeting a unique feature of tissue-resident memory CD4+ T cells in lungs.
IL-25-responsive migratory iILC2s require BATF and stably convert to tissue-resident nILC2s
Authors: Miller Mindy M , Danhorn Thomas , Reinhardt R. Lee
Published: January 2023

Inflammatory ILC2s (iILC2s) are a transient subset of group-2 innate lymphoid cells that migrate to the lung shortly after helminth infection or IL-25 administration. Our recent findings identified the AP-1 factor BATF as the major transcriptional regulator required for iILC2 generation and function. Batf-deficient mice infected with the helminth Nippostrongylus brasiliensis display a selective defect in IL-25-mediated worm clearance and a loss of iILC2s but not nILC2s. This corresponds with the absence of early cytokine production by ILC2s in Batf-deficient mice, indicating the requirement of this transcription factor in ILC2 function. The fate of iILC2s is currently unknown as this subset of ILC2s is found only transiently in the lung following infection. Taking advantage of our finding that Arginase-1 (Arg1) expression can delineate tissue-resident nILC2 (Arg1+) and migratory iILC2 (Arg1−) populations, we used an Arg1 reporter mouse to address whether iILC2s could acquire an Arg1+ nILC2-like phenotype. Upon transfer into a congenic host, sorted Arg1− iILC2s acquired Arg1 expression and further adopted characteristics of nILC2s. These converted cells stably persisted after transfer indicating a potential long-term contribution of migratory iILC2s to the tissue-resident nILC2 pool in the lung.
IL-7 shapes a multifaceted CD8 T cell response to airway Influenza/A infection
Authors: Sheikh Abdalla A , Jackson Jennie , Shim Brian , Plumb Adam , Abraham Ninan
Published: January 2023

The lungs are a site vulnerable to diseases such as cancer, autoimmunity and infections. With each breath, we risk inhaling infectious agents, and as such, airborne diseases are the leading cause of infectious disease-related deaths in the world. In particular, various strains of Influenza virus can infect airway epithelial cells and activate a network of immune cells leading to clearance, or in cases of pandemic strains, an overzealous response that can be fatal.

Adaptive immune cells, particularly cytotoxic CD8 T cell lymphocytes, play a crucial role in controlling viral replication by killing infected cells. Therefore, tight regulation of the cytokines that lead to the proper activation, proliferation and function of these immune cells is necessary to clear infections efficiently while minimizing damage to the host. Interleukin-7 (IL-7) is a cytokine known for its importance in T cell development and survival. While the function of IL-7 in T cell survival is well characterized, how IL-7 shapes T cell effector responses when a pathogen is encountered is less understood.

Using IL-7Rα hypomorph mice in chimeric and adoptive transfer experiments we have found that IL-7 is cell-intrinsically important for the priming of antigen specific CD8 T cells in the draining lymph nodes. We found that IL-7 dictates terminal differentiation, cytokine production and degranulation of CD8 T cells locally in the airways. Drugs that manipulate IL-7 signaling are currently under clinical trial for multiple conditions. Our findings on IL-7 and its effects on lower respiratory diseases will be necessary for expanding the utility of these therapeutics.
Shedding LIGHT and TL1A on fibrosis
Authors: Herro Rana , Croft Michael
Published: January 2023

Anti-inflammatory drugs have so far failed to reduce fibrosis and tissue remodeling in the clinic. Alternatively, 5 TNF inhibitors have been FDA approved to treat different inflammatory diseases and are safe to administer to patients. In human, LIGHT and its receptors have been linked to numerous autoimmune and inflammatory diseases with fibrotic components. We show that blocking LIGHT signaling in isolation is efficient in reducing fibrosis in the skin and lung of mice induced with bleomycin to mimic human SSc and IPF, as well as in 2 severe allergen-driven models of asthma and atopic dermatitis. Neutralizing LIGHT signaling through its receptors by genetic deletion or antibody blocking, dramatically decreased fibrosis in the lungs and skins. Moreover the specific deletion of LIGHT-receptor HVEM on keratinocytes or the therapeutic blocking of LIGHT-HVEM interaction post-disease onset, abrogated skin fibrosis. LIGHT seems to play a central role since it can control the expression of major pro-fibrotic factors such as TSLP, IL-13, TGF-b and Periostin. Also LIGHT alone can induce a fibro-proliferative disorder that mimics human SSc, when administered alone. Lastly we observed an upregulation of LIGHT and its receptors in lung biopsies of patients suffering from asthma, SSc and IPF. More recently, we discovered another TNF family Member called TL1A can also potentiate fibrosis similarly to LIGHT and which signaling can promote tissue remodeling. Using antagonistic fusion proteins to neutralize LIGHT and TL1A signaling, we treated skin fibrosis and observed greater efficacy when LIGHT and TL1A were targeted concomitantly. The relevance of this work to fibrosis therapies associated with asthma, AD, SSc and IPF are tremendous.
The role of RUBCN in exacerbating allergic airway inflammation
Authors: Martinez Jennifer , Oguin Thomas H , Kolb Joseph P , Whitehead Gregory , Thomas Seddon Y , Muse Ginger W , DeGraff Laura Miller , Cook Donald N.
Published: January 2023

Allergic airway inflammation (typified by atopic asthma) is a classic example of type 2 immunity, which can be triggered by non-microbial environmental stimuli, such as allergens. Importantly, type 2 immunity engages the entire affected tissue and can involve the activity of not only immune cells, like Th2 cells and eosinophils, but also non-hematopoietic cells, like epithelial cells. Often, type 2 immunity exists in competition with type 1 immunity, classically defined as the activity of phagocytic cells, such as classically activated “M1” macrophages, and IFNg-producing adaptive immune cells, like CD8+ T cells and Th1 cells. Our past studies have demonstrated that deficiency in LC3-associated phagocytosis (LAP), a non-canonical form of autophagy requiring Rubcn, results in increased type 1 inflammation across a variety of stimuli, suggesting that LAP represents an important mechanism to regulate the inflammatory response. How Rubcn deficiency affects the onset and severity of type 2 immunity, such as allergic airway inflammation, remains unknown. Here, we demonstrate that, in response to house dust extract (HDE)/OVA exposure, lungs from Rubcn−/− mice contain increased levels of Th1 and Tr17 cells (potent regulatory CD4+ T cells that co-express FOXP3 and RORg), decreased Th2 cells, and decreased neutrophils, resulting in overall protection from allergic airway pathology. Rubcn-deficiency was required in non-hematopoietic cells, presumably airway epithelial cells, to exert this protective effect. As approximately 50% of asthmatics suffer from corticosteroid-resistant, neutrophil-mediated inflammation, inhibition of RUBCN could represent a novel therapy for steroid-resistant asthmatics.
Zom-biecoming: Single-Cell RNA-sequencing reveals senescence-like features of alveolar macrophages during aging
Authors: Wu Yue , Zhu Bibo , Zhang Ruixuan , Goplen Nicholas PJ. , Wang Zheng , Li Ying , Cardani-Boulton Amber , Braciale Thomas J , Sun Jie
Published: January 2023

Alveolar Macrophages (AMs) are unique innate immune cells that reside in the alveolar space and accommodate the ever-changing need of the lungs against internal and external challenges. During homeostasis, AMs maintained themselves through self-renewal without the need for input from adult hematopoietic stem cells. Currently, little is known about how aging influences AM dynamics, heterogeneity and self-renewal. To this end, we have performed single-cell RNA sequencing (scRNA-seq) on murine lung AMs (CD11+ Siglec F+) isolated from young (~8w) and aged (~2y) mice. We discovered that despite similar transcriptome in the proliferating cells, AMs from aged mice had reduced proliferation and self-renew ability than those from the young mice. AMs from aged mice also displayed elevated senescence markers such as p16, p21 and p53. Consistently, AMs from aged mice had reduced DNA repair ability, which could contribute to their impaired capacity to pass through the cell cycle checkpoints. Interestingly, AMs from aged mice also possessed reduced pro-apoptotic gene profile. Thus, we hypothesize that the reduced proliferation- and apoptosis-associated gene profiles in aged AMs may lead to the enhanced senescence-like phenotype in those cells. Further validation of the senescence-like phenotype of aged AMs and investigation of how they contribute to lung “inflammaging” are warranted.
Natural killer cells orchestrate angiogenesis
Authors: Wolf Katharine , Bilal Mahmood , Schneiderman Sylvia , Dambaeva Svetlana , Beaman Kenneth D
Published: January 2023

Tumor-associated angiogenesis is accompanied by immunosuppression mediated by tumor cells and the immune microenvironment. Similarly, embryo implantation in the uterus requires both angiogenesis and immunosuppression. Uterine natural killer (NK) cells promote immune tolerance for the allogenic invasion of the embryo during pregnancy. In these studies, we explore the mechanism of NK cell-mediated vascular remodeling. RNA sequencing demonstrates a subpopulation of uterine NK cells that are enriched for proangiogenic genes in the uterus during pregnancy. Ephrin-B2 is a receptor tyrosine kinase well known for promoting angiogenesis in cancer, however, the mechanism for ephrin-B2-mediated angiogenesis in the uterus is not known. NK cells were isolated from the uterus and spleen of mice. Isolated NK cells were characterized for the expression of ephrin-B2 by immunofluorescence and flow cytometry. We show that uterine, but not splenic, NK cells express ephrin-B2. Because uterine NK cells also mediate immunosuppression, uterine NK cells were probed for expression of cytotoxicity markers. Cytotoxic marker CD27 is significantly upregulated in conventional NK cells, but not tissue-resident NK cells. Our data show that tissue resident uterine NK cells express pro-angiogenic marker ephrin-B2 and that these cells are not cytotoxic. This describes a previously unsuspected function for NK cells.
Comparison of the therapeutic effects between topical 8-oxo-2′-deoxyguanosine and steroid in ocular chemical burn experimental model
Authors: Kim Dong Hyun
Published: January 2023

Purpose

To compare the effects of topical 8-oxo-dG and steroids on ocular chemical burn mice model

Methods

We created ocular chemical burn models with 8-week-old BALB/c mice (n=45) by applying 0.1M sodium hydroxide(NaOH); the mice were then treated with 8-oxo-dG eye drops 10 mg/ml, 0.1% fluorometholone ophthalmic suspension, and phosphate buffered saline (PBS) twice daily. After seven days, clinical findings such as corneal integrity, clarity, and neovascularization were assessed. Histology, immunohistochemistry, inflammatory cytokine levels using real time polymerase chain reactions (PCRs), and amount of total reactive oxygen species in the corneas of the mice were also compared.

Results

Topical application of 8-oxo-dG and steroid resulted in a significant improvement of epithelial defects and clarity, compared to PBS (each p<0.05). Stromal thickness in cornea was also decreased in the 8-oxo-dG and steroid treated mice, compared to PBS-treated controls. (p<0.001) The expressions of F4/80 and neutrophil elastase-positive inflammatory cells in the stroma, and IL-1β and IL-6 cytokine levels in whole cornea were significantly reduced in the 8-oxo-dG group, compared to PBS group (each p<0.05).

Conclusion

In the ocular chemical burn experimental model, topical 8-oxo-dG showed excellent anti-inflammatory effects those were not inferior than topical steroids.
Low dose interleukin-2 decreases P-glycoprotein activity and exerts synergistic anti-inflammatory effects with immunosuppressants in the treatment of lupus nephritis with steroid and immunosuppressive drug resistance
Authors: Wang Meiying , Hu Yiping , Yu Shengyan , Wang Qingwen , Singh Ram Raj
Published: January 2023

Objective

P-glycoprotein (P-gp) is capable of transporting glucocorticoids and immunosuppressants out of lymphocytes, resulting in drug resistance in lupus nephritis (LN). IL-2 decreases P-gp expression in cultured cells from human colon carcinoma. Here, we investigated the effects of low-dose IL-2 on the functionality of P-gp in PBMCs of steroid resistant LN patients and in kidneys in MRL/lpr mice.

Methods

30 drug resistant LN patients were treated with glucocorticoids and immunosuppressants with or without low-dose IL-2 for 12 weeks. Serum and urine collected before and after treatment were analyzed for anti-dsDNA, C3, C4, renal and liver function. P-gp expression and activity were detected in PBMCs by flow cytometry and rhodamine 123. MRL/lpr mice were treated with methylprednisolone (MP) with or without IL-2 for 4 weeks. Pathological changes as well as protein and mRNA expression of P-gp were assessed in kidneys.

Results

Compared to patients without IL-2 treatment, the patients treated with IL-2 had higher serum C3 (p=0.01) and C4 levels (p=0.04), while lower proteinuria(p=0.04), serum creatinine (p=0.03), anti-dsDNA levels (p< 0.001), and accordingly lower SLEDAI (p=0.03). These modifications were associated with reduced P-gp expression (p< 0.01) and activity (p=0.02) in PBMCs. Moreover, compared with controls, MRL/lpr mice treated with IL-2 and MP showed reduced proteinuria (p=0.002) and fewer acute and chronic renal pathological lesions. IL-2 attenuated renal deposition of IgG/C3, decreased renal expression of P-gp.

Conclusion

Low-dose IL-2 decreases P-gp activity and exerts synergistic anti-inflammatory effects with immunosuppressants in the treatment of LN with steroid and immunosuppressants resistance.
Response Gene to Complement -32 facilitates local recruitment of IL-17-producing cells in immune complex mediated glomerulonephritis through the CCR6/CCL20 axis
Authors: Rus Violeta , Tatomir Alexandru , Nguyen Vinh , Papadimitriou John , Drachenberg Cinthia , Sung Sun-Sang J , Fu Shu Man , Luzina Irina G , Badea Tudor Constantin , Rus Horea G
Published: January 2023

Response Gene to Complement (RGC)-32 is a cell cycle regulator induced by complement activation, growth factors and cytokines. RGC-32 mediates TGF-β dependent profibrotic pathways, while in immune cells promotes the differentiation of Th17 cells. In the nephrotoxic nephritis (NTN) mouse model of glomerulonephritis (GN), RGC-32 promotes organ damage through proinflammatory and profibrotic effects. To define the mechanisms underlying the role of RGC-32 in immune complex GN, we induced NTN in WT and RGC-32−/− mice. Proteinuria, blood urea nitrogen and kidney histopathology were determined to assess kidney damage. Single cell suspension of infiltrating kidneys and spleens cells were analyzed by flow cytometry and PCR for expression of IL-17A, CCR6, CCL20, CXCL1, CXCL2 and CXCL5.

Induction of NTN in RGC-32−/− mice resulted in decreased expression of IL-17A and CCR6 mRNA and decreased proportion of renal IL-17A+ CD4 T cells. The decrease in IL-17A+ CD4 cells was paralleled by decreased mRNA expression of CCL20 in both the renal tissue and infiltrating kidney cells of RGC-32−/− mice. In vitro, CD4+ T cells from spleens of RGC-32−/− mice cultured under Th17 conditions displayed decreased expression of CCL20 mRNA. These data suggest a role for RGC-32 in the positive feedback loop that enhances Th17 cell recruitment. The decrease in renal IL-17A+ cells was associated with decreased expression of IL-17 induced chemokines CXCL1, CXCL2 and CXCL5 and decreased local infiltration of neutrophils. These results suggest that RGC-32 promotes the proinflammatory pathway in immune complex mediated GN by enhancing local recruitment of IL-17A+ cells through the CCR6/CCL20 axis. RGC-32 is a potential novel therapeutic target in the treatment of LN.
IL-35 producing antigen presenting cells in type 1 diabetes
Authors: Luo Zhengkang , Blixt Martin , Carlsson Per-Ola , Espes Daniel , Sandler Stellan , Singh Kailash
Published: January 2023

In type 1 diabetes (T1D), the insulin producing β cells are damaged by unknown factors. Antigen presenting cells (APCs) are believed to present autoantigens to T cells, thus initiating the immune attacks to β cells. However, several types of APCs also possess regulatory functions by producing anti-inflammatory cytokines like IL-35. We found the proportions of IL-35+ cells among both CD11c+CD123− dendritic cells (DCs) and CD11c−CD123+ DCs were decreased in the peripheral blood mononuclear cells from T1D patients than healthy controls. Multiple low dose streptozotocin (MLDSTZ) mouse model of T1D was used to study the immune response of IL-35 producing APCs. Male CD-1 mice were injected with STZ for five consecutive days to induce experimental T1D. We found the proportions of IL-35 expressing MHC-II+CD11b−CD11chigh conventional DCs (cDCs) were decreased in the spleens of MLDSTZ mice than in control mice on day 21 after the first STZ injection. We next investigated which subtype of cDCs contribute to the IL-35 production, and found that it is CD8+XCR1+ cDC1s that produce IL-35 while cDC2s do not. After the five day STZ injections, male CD-1 mice further received recombinant mouse IL-35 injection or PBS daily for 8 days. We found increased proportions of IL-35+ cells among cDC1s in IL-35 treated mice than PBS treated mice. Only a minority of plasmacytoid DCs (pDCs) produce IL-35, and IL-35 treatment did not increase the proportion of IL-35+ pDCs. We further investigated macrophage polarization, finding that IL-35 treatment increased Arg1+ M2 proportions and decreased TNF-α+ M1 proportions. Our results illustrate that the decrease in IL-35 producing APCs may contribute to the development of T1D.
Iloprost-treated dendritic cells promote antigen-specific regulatory T cell differentiation in mice
Authors: Wong Tzu-Hsuan , Gau Rong-Jiun , Chen Yu-Fang , Shen Hsin-Hsin , Lin Carl Tsai-Yu , Chen Sen-Lu , Suen Jau-Ling
Published: January 2023

Iloprost, a stable prostaglandin I2 (PGI2) analog, can inhibit allergic inflammation in an OVA-induced asthma model via inhibition of airway dendritic cell (DC) function. However, the underlying mechanism of PGI2 signaling-mediated immunosuppression remains unclear. This study explored whether iloprost-treated DCs can suppress inflammation by promoting antigen-specific regulatory T cell (Treg) differentiation. We established an allergic lung inflammation model using a hydrogel biomaterial delivery system and observed that iloprost significantly suppressed OVA-induced Th2 lung inflammation and increased the frequency of OVA-specific Tregs in vivo. We further observed that iloprost-treated DCs displayed tolerogenic characteristics, including low inflammatory cytokine (IL-12p70, TNF-α, IL-6, IL-23) expression levels, high anti-inflammatory cytokine (IL-10) production, and a semimature phenotype. In addition, iloprost-treated DCs increased OVA-specific CD4+Foxp3+ T cell differentiation from naïve T cells in vitro and in vivo. Blocking experiments showed that iloprost-treated DCs promoted Treg differentiation, at least in part, through PD-L1. Furthermore, iloprost treatment suppressed DC-mediated airway inflammation and increased the frequency of OVA-specific Tregs through PD-L1 in vivo. Taken together, these results show that PGI2 signaling in DCs may lead to immune tolerance, suggesting that the PGI2 analog has the potential to be applied therapeutically for tolerogenic DC immunotherapy in autoimmune diseases or allergic asthma.
Olfactory ecto-mesenchymal stem cells derived exosomes reverse the immunosuppressive capacity of myeloid-derived suppressor cells to ameliorates experimental Sjögren’s syndrome
Authors: Rui Ke , Tian Jie , Hong Yue , Lu Liwei , Wang Shengjun
Published: January 2023

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease characterized by progressive inflammation and tissue damage of salivary glands and lacrimal glands. Our previous studies have shown myeloid-derived suppressor cells (MDSCs) were significantly increased but exhibited gradually diminished suppressive capacity in experimental Sjögren’s syndrome (ESS), thus leading to the development of the disease. In this study, we found that exosomes derived from olfactory ecto-mesenchymal stem cells (OE-MSCs-Exo) effectively enhanced the suppressive function of MDSCs on T cell proliferation, up-regulated MDSC-mediated suppressive factors (Arginase, ROS and NO), and subsequently suppressing the development of ESS. Additionally, levels of CD40, CD80, CD86 and MHCII on MDSCs were dramatically decreased, indicating the differentiated late stage MDSCs were reversed to the original undifferentiated status by OE-MSCs-Exo. Further investigation demonstrated that the increased suppressive function of MDSCs was orchestrated by IL-6, from both exosomes secreted IL-6 as well as its own IL-6 autocrine signaling pathway. Taken together, OE-MSCs-Exo have the potential to alleviate ESS severity through inducing MDSC expansion and potentiating their immunosuppressive function, indicating OE-MSCs-Exo may represent a new therapeutic strategy for the treatment of Sjögren’s syndrome and other autoimmune diseases.
IL-37 increases liver inflammation in Con A-induced hepatitis by increasing IFN-γ secretion of infiltrated NK Cells
Authors: Lin Chia I , Tsao Cheng Chiu , Chuang Ya Hui
Published: January 2023

Liver is an immunologically tolerogenic organ, while, autoreactive immune cells mediated autoimmune hepatitis (AIH) is observed. Suppression of the hyper-reactive immune responses may restore the tolerance of liver. IL-37, an emerging IL-1 family cytokine with anti-inflammatory effects on innate and adaptive immunity, shows benefit on many autoimmune diseases. We proposed IL-37 could inhibit immune responses and improve AIH. IL-37 expressing adeno-associated virus (AAV-IL-37) successfully dampened the activation of macrophages in vitro. We next analyzed the effect of IL-37 in Con A-induced autoimmune hepatitis mouse model. Surprisingly, the liver histopathology showed more necrotic hepatocytes and infiltrating immune cells in AAV-IL-37 treated Con A mice than in control mice. Higher levels of serum IFN-γ in IL-37-expressing Con A mice were observed. Moreover, expression of inflammation-related mRNA in the liver were also highly induced with AAV-IL-37 treatment. Among hepatic lymphoid cells, NK and NKT cells were particularly increased in IL-37 treated Con A mice. The frequency of IFN-γ-secreting NK cells was also significantly increased. Further, CCL5, a chemoattractant for NK cells, was significantly elevated in the serum of AAV-IL-37 injected Con A mice. These results suggest that IL-37 worsen liver inflammation instead of relieving the disease. Of note, there was no obvious inflammation in AAV-IL-37 treated naïve mice, suggesting the proinflammatory effect of IL-37 in Con A treated mice resulted from the inflamed liver microenvironment. In conclusion, IL-37 aggravated liver inflammation in Con A induced hepatitis through recruiting NK cells and elevating the IFN-γ secretion of NK cells.
Single-cell analysis of autoreactive B cells in patients with systemic sclerosis revealed that responses to therapy are determined by effector and regulatory B cell balance
Authors: Ebata Satoshi , Yoshizaki Ayumi , Fukasawa Takemichi , Sato Shinichi
Published: January 2023

Objective

Systemic sclerosis (SSc) is a connective tissue disease with poor prognosis. Although the standard treatment for skin and lung fibrosis is cyclophosphamide (CYC), there are not a few non-responders to the treatment. Recent studies showed that autoreactive B cells play a critical role in SSc. We hypothesized that these B cells determine the effectiveness of CYC treatment. We focused on cytokine profiles of single autoreactive B cells in patients with SSc.

Methods

We simulated the interaction of B cells and endothelial cells in microfluidic cell culture systems. We cultured endothelial cells in a microchannel. Then, we loaded B cells of patients with SSc. We collected single B cells which adhere to endothelial cells by trypsin treatment. We stimulated these B cells, with PMA and ionomycin, and measured the levels of produced interleukin (IL)-10 and IL-6 to detect the percentages of regulatory and effector B cells, by our original micro fluidic-ELISA system.

Results

In SSc patients, the number of B cells adhering to endothelial cells significantly increased compared with healthy controls. Most of these B cells produced anti-endothelial cells antibodies. The frequencies of IL-10-producing regulatory B cells after the CYC treatment increased in responders and decresed in non-responders. Those of IL-6-producing effector B cells after the treatment decreased in responders and increased in non-responders.

Conclusions

The effectiveness of CYC treatment to SSc patients is associated with cytokine profiles of B cells, which interact with endothelial cells. Checking the profiles of these B cells, after the first few months on from initiation of CYC therapy, may help to decide whether or not we should continue the treatment.
Enhancing ADCC by human natural killer cells to improve tumor cell killing
Authors: Snyder Kristin M , Hullsiek Robert , Mishra Hemant Kumar , Dixon Kate J , Mendez Daniel C , Li Yunfang , Rogich Allison , Wu Jianming , Walcheck Bruce
Published: January 2023

Natural killer (NK) cells are cytotoxic lymphocytes that can rapidly kill tumor cells with exquisite specificity by antibody-dependent cell-mediated cytotoxicity (ADCC), which is exclusively mediated by the IgG Fcγ receptor CD16A. Many clinically successful therapeutic monoclonal antibodies (mAbs) utilize ADCC as a mechanism of action. However, CD16A binds IgG with low affinity and is rapidly cleaved by a proteolytic process from the cell surface upon activation, limiting therapeutic mAb efficacy. We generated NK cells expressing engineered high affinity FcγRs. Here, we investigated CD64/16A, a recombinant receptor consisting of the extracellular region of CD64, the highest affinity IgG FcγR, and the transmembrane and intracellular regions of CD16A. We expressed CD64/16A in human NK92 cells, which lack endogenous FcγRs but mediates ADCC upon expression of CD16A, and in induced pluripotent stem cells differentiated into NK (iNK) cells. We determined that CD64/16A was functional in vitro, facilitated ADCC and the production of cytokines, did not undergo rapid downregulation in expression upon NK cell activation, and facilitated conjugation to antibody-opsonized target cells. We are currently exploring an in vivo xenograft model using NSG immunocompromised mice engrafted with a HER2+ ovarian cancer cells to further examine the ADCC potency of CD64/16A iNK cells. Our findings suggest that CD64/16A may be utilized by engineered NK cells, leading to the formation of an “off-the-shelf” cellular therapy that can be combined with therapeutic mAbs for the treatment of various tumor types.
Targeting REGNASE-1 improves efficacy of chimeric antigen receptor T cell therapy for leukemia
Authors: Zheng Wenting , Wei Jun , Jones Lindsay , Chi Hongbo , Geiger Terrence
Published: January 2023

Chimeric Antigen Receptor (CAR) modified T cells have been successful in treatment of acute lymphoblastic leukemia (ALL), however the efficacy of CAR T cell therapy is often limited by the poor persistence and function of adoptively transferred cells. We identified REGNASE-1 as a negative regulator of antitumor responses that modulates the persistence and effector function of adoptively transferred CD8+ T cells. [1] We developed a novel model to study the efficacy of Regnase-1-deficient CAR T cells in an immune replete environment. T cells from transgenic mice expressing a clinically relevant anti-human CD19 CAR were used to treat primary mouse ALL cells that express human CD19. We find that REGNASE-1-deficient CD8+ CAR T cells have improved in vivo persistence and exhibit markedly improved therapeutic efficacy against ALL. REGNASE-1-deficient CD8+ CAR T cells undergo antigen-dependent reprogramming to a naïve/memory-like phenotype, with simultaneous antigen-independent reduction in exhaustion marker expression. Regnase-1-deficiency also leads to enhanced memory responses of CAR T cells after primary tumor clearance. Our findings indicate that Regnase-1 modulates long term persistence and memory in CAR T cells. Depletion of Regnase-1 provides a new approach to improve the therapeutic efficacy of CAR T cell therapy.

Supported by ALSAC/St. Jude Children’s Research Hospital and The Assisi Foundation of Memphis
Fasting Mimicking Diet promotes immunotherapy-associated changes and inhibits autophagy to promote cancer free survival in Acute Lymphoblastic Leukemia models
Authors: Buono Roberta , Tucci Jonathan , Cutri Raffaello , Guidi Novella , Mangul Serghei , Pellegrini Matteo , Fruman David A , Mittelman Steven D. , Longo Valter
Published: January 2023

One of the new therapeutic strategy to promote cancer free survival is to combine dietary intervention like fasting or fasting mimicking diets (FMDs) with cancer treatments. FMDs enhance the efficacy of a wide variety of therapies ranging from chemotherapy to kinase inhibitors to immunotherapy, weakening cancer cells while protecting normal cells and tissues.

In this work we show that FMD in combination with vincristine (VC), enhance the death of acute lymphoblastic leukemia (ALL) by inhibition of autophagy and induction of apoptosis by p53 modulation and activation of caspase-3. Our data indicates that a periodic FMD increases the efficacy of VC and extends the cancer free survival of an in-vivo mouse syngeneic pre-B ALL model. We identify in human and mice leukemic cells that the autophagy inhibition is the mechanism used by FMD to potentiate the cytotoxic effect of VC. Finally, in vivo treatment of FMD+VC with an autophagy inhibitor shows a synergistic effect on increasing survival in ALL mice.

Furthermore, FMD induces activation of anticancer immunity. The gene expression profile consistent with anti-cancer immune responses resulting from VC plus FMD together with accelerated cancer progression when these treatments were combined with anti-CD8 antibodies indicate a central role of the immune system in these effects. Thus, the inhibition of autophagy and enhancement of immune responses are responsible for the ability of FMD to promote cancer free survival in ALL mice.
Macrophage repolarization via leptin increases immunotherapy efficacy in obesity
Authors: Dudzinski Stephanie O , Beckermann Kathryn E. , Young Kirsten , Hongo Rachel , Giorgio Todd , Rathmell Jeffrey
Published: January 2023

Immune checkpoint blockade treatment is life-saving for some patients, but most do not respond. While systemic immune dysfunction occurs in obesity, early studies have surprisingly shown that obesity increases immunotherapy efficacy. Obesity promotes immune dysfunction through T cell exhaustion, which is well understood, but obesity also affects macrophage polarization, which is understudied in the setting of obesity, cancer, and immunotherapy. Male C57BL/6 mice received a 60 kcal high-fat diet for 12 weeks and received 250,000 MC38 colon cancer cells subcutaneously. Mice received 200 ug of IgG control antibody or anti-PD-1 antibody every 2 days starting on day 5. Tumors were collected 16 days post-injection and processed for flow cytometry. Samples were stained for T cells (CD4+ helper, T Regulatory, and CD8+ cells), macrophages (M1 and M2), and myeloid derived suppressor cells. Obese mice had larger tumor growth rates and tumor burden than lean mice. Tumors in obese mice had fewer CD8+ and CD4+ T cells, but the T cells expressed higher levels of CD44 and PD1. MC38 tumors in obese mice had greater immunotherapy efficacy than lean mouse tumors. Flow cytometry revealed that the anti-PD-1 treatment repolarized the macrophages to an M1-phentoype, whereas anti-PD-1 treatment in lean mice increased the T cell percentage in tumors. In experiments where lean mice received leptin treatments to emulate the elevated leptin in obesity, leptin-treated mice had smaller tumors and an increased M1-phenotype of macrophage. Our work shows that macrophage repolarization was the most significant immune cell change in our obesity and immunotherapy studies. Future work will investigate the efficacy of immunotherapy in macrophage depleted models.