bluuebunny/crossref_metadata_2025 huggingface dataset:
An 18-day Residential Lifestyle Program Improves Fitness of Asthma Patients
Authors: Ramirez Francisco E , Nedley Neil , Freed Spencer
Published: January 2023

Rationale

Lifestyle interventions are known to improve many medical conditions. We are documenting the effect that a, 18-day residential medical lifestyle program has on the fitness of asthma patients using metabolic equivalent (MetS), which correlate to exercise capacity.

Methods

The program included 3 daily buffet-like whole food plant-based meals with no snacks, 2 to 3 times a day sessions of exercise (aerobic and anaerobic), massage, hydrotherapy, medical and spiritual interventions. The program is called NEWSTART program and takes place in Weimar, California. NEWSTART means: Nutrition, Exercise, Water, proper Sunlight exposure, fresh Air, Rest and Trust (relational, spiritual, and psychological aspects). Board certified physicians monitored patient progress. Nutritionist, exercise physiologist, psychological, and chaplaincy care is also provided. From 2282 patients that completed the 18-day NEWSTART program in 13 years we found 66 that had the starting diagnosis of asthma. From those 66, 39 had a pre and post stress test to determine MetS. These 39 patients had an average age of 59, SD 14.9.

Results

At baseline, asthmatics had on average a MetS of 8.38, SD 3.3, median 9, mode 10, min 2 and max 16. At the end of the program the stress test reported a MetS of 9.6, SD 3.8, median 10, mode 9, min 2 and max of 18. Asthmatic crises decreased during the program.

Conclusions

The lifestyle Program seems to improve fitness of asthma patients. The end MetS of 9.6 is important since a 10 to 11 MetS is considered good exercise capacity and may decrease mortality.
Experimental infection with human rhinovirus-A16 reveals an amplified Th1 response in allergic asthmatics
Authors: Muehling Lyndsey , Agrawal Rachana , Wright Paul , Kwok William , Heymann Peter , Woodfolk Judith
Published: January 2023

Human rhinovirus (RV) is a major cause of the common cold and is known to induce acute asthma episodes in allergic subjects. Despite established roles for CD4+ T cells in both anti-viral immunity and allergic disease, the T cell mechanisms of RV-induced asthma are poorly understood. Current theory suggests a role for deficient anti-viral responses, as well as pro-allergic Th2 responses. In this study, we leveraged new pMHCII tetramer reagents to specifically probe both RV- and allergen-specific circulating CD4+ T cell responses to experimental RV infection in allergic asthmatic and control subjects. T cells were monitored for 3 weeks to capture acute and convalescent phases. RV-specific cells were readily detected prior to infection in both asthmatics and controls, whereas allergen-specific cells were only detected at appreciable frequencies in asthmatic subjects. While allergen-specific cells displayed predominantly Th2 phenotypes (CCR4+, CRTH2+) in allergic asthmatics, RV-specific cells were Th1-like (CXCR3+, CCR5+). The RV-specific Th1 signature was enhanced during acute infection and persisted into convalescence. A minority of cells of both specificities expressed hybrid Th1/Th2 molecular signatures. As predicted, RV-specific cells expanded and became activated during infection with RV-A16. However, this response was augmented in allergic asthmatics, and was accompanied by increased numbers of allergen-specific cells. Additionally, plasmacytoid dendritic cells (pDCs) increased during acute infection, but only in asthmatics. Taken together, these data suggest that RV-induced asthma is characterized by an augmented anti-viral Th1 response that is mediated by pDCs, and which co-opts allergen-specific Th2 cells.
Systemic markers of inflammation and neuronal injury in patients with epilepsy
Authors: Kunda Shailaja , LaFrance-Corey Reghann G. , Khadjevand Fatemah , Worrell Gregory A , Howe Charles L
Published: January 2023

Epilepsy is a chronic neurologic disorder characterized by recurrent seizures and variable cognitive and neurodevelopmental issues. In many individuals the underlying pathogenesis is unknown, but burgeoning evidence indicates a link between neuroinflammation and seizure development. Therapeutically targeting inflammation in epilepsy is of particular interest for patients that do not respond to the current standard of care. Likewise, identifying changes in peripheral immune status may provide a unique biomarker for seizure prediction. Using high-resolution repeated measures temporal profiling of serum cytokine levels and flow cytometric analysis of the activation status of circulating neutrophils and monocytes, we established an immune profile for patients undergoing multi-day continuous video-EEG monitoring. We identified an apparent temporal correlation between clinical seizure events and increased levels of serum CCL2, as well as evidence of acute peri-ictal changes in the status of inflammatory monocytes (CD14++CD16+ and CD14+CD16++) in 4 patients. We also measured acute peri-ictal changes in a marker of neuronal injury in these patients. Ongoing studies will correlate patient immune status with neuronal injury markers and attempt to incorporate analysis of sub-clinical EEG changes with the immunophenotype.
Multispecific recognition of developmental neuronal antigens and neurite outgrowth inhibition by an anti-myelin proteolipid protein (PLP) peptide monoclonal antibody (mAb)
Authors: Sobel Raymond A , Hinojoza Julian R , Eaton Mary Jane
Published: January 2023

CNS injury results in the generation of antibodies to self-antigens, including epitopes of PLP, the most abundant CNS myelin protein. We previously found that despite similar recognition of CNS vertebrate myelin, anti-PLP50–69 mAbs F4.4C2 (IgG1κ) and F3.6E9 (IgG2aκ) differ in their recognition of neurons (Greenfield et al, 2006). To identify the neuronal mAb targets and determine the potential pathobiological significance of this finding, we used rat hippocampal precursor cells (RHPC) and PC12 cells, a nerve growth factor (NGF)-responsive neuronal cell line that does not express PLP. Both mAbs bound RHPC and PC12 cells and inhibited their in vitro neurite outgrowth; they differed in recognition of extracted PC12 cell proteins on Western blot and in extent of their recognition of human CNS neurons of different ages and in adult neural stem cell niches (NSCN). Thus, epitope sequence alone did not predict mAb neuronal reactivity. We immunoprecipitated proteins from NGF-treated and -untreated PC12 cell extracts using F4.4C2- and F3.9E9-coated magnetic beads; SDS-PAGE-separated bands of eluates from the beads were cut from gels and analyzed by GeLC-MS/MS. mAb F4.4C2 consistently captured the proteolipid gene family M6 neuronal protein. M6b and PLP50–69 have 45% sequence identity. M6b is required for neurite outgrowth and axon guidance and is expressed in developing human CNS neurons and NSCN. F4.4C2 also immunoprecipitated EphA family proteins and other cell surface growth and differentiation molecules expressed in human NSCN. Thus, certain anti-PLP autoantibodies generated after myelin injury may inhibit neuronal repair in the developing and adult human CNS by multispecific binding to neuronal growth and differentiation molecules.
Immune response in Parkinson’s disease driven by HLA display of α-synuclein peptides
Authors: Arlehamn Cecilia S Lindestam , Alcalay Roy N. , Garretti Francesca , Cote Lucien , Kanter Ellen , Agin-Liebes Julian , Liong Christopher , McMurtrey Curtis , Hildebrand William H , Mao Xiabo , Dawson Valina , Dawson Ted M , Oseroff Carla , Pham John , Sidney John , Dillon Myles , Carpenter Chelsea , Weiskopf Daniela , Phillips Elizabeth J , Mallal Simon Alexander , Peters Bjoern , Frazier April , Sulzer David , Sette Alessandro
Published: January 2023

Abnormal processing of self-proteins can produce epitopes presented by major histocompatibility (MHC) proteins to be recognized by specific T cells that escaped central tolerance during thymic selection. Such actions by the acquired immune system are widely held to produce autoimmune disorders such as Type-1 diabetes. While not considered to possess autoimmune features, neurodegenerative diseases are characterized by the altered processing of specific proteins. One of the major pathological features of Parkinson’s disease are the presence of intraneuronal aggregrates known as Lewy bodies and neurites composed of α-synuclein. Genetic studies associate Parkinson’s disease with DRB5*01 and DRB1*15:01 MHC alleles. To address the hypothesis that Parkinson’s disease is associated with T cell recognition of epitopes derived from α-synuclein and presented by specific MHC alleles, we recruited 67 Parkinson’s disease patients and 36 age-matched non-Parkinson’s healthy controls. We found that a defined set of peptides derived from α-synuclein, act as antigenic epitopes displayed by these alleles and drive helper and cytotoxic T cell responses in Parkinson’s disease patients. We identified two antigenic regions in α-synuclein, the first near the N terminus (aa31-46), which was bound by DRB1*15:01 and DRB5*01:01, and the second near the C terminus, which required phosphorylation of an amino acid residue (S129). These α-synuclein epitopes were shown to arise from natural processing of both extracellular native α-synuclein and the fibrilized form associated with Parkinson’s disease pathogenesis. These responses may explain the association of Parkinson’s disease with alleles of the acquired immune system.
The transcription factor basic helix loop helix, member e40 is required for establishment of proper peritoneal macrophage identity
Authors: Jarjour Nicholas N , Lin Chih-Chung , Bradstreet Tara R , Schwarzkopf Elizabeth A , Huang Stanley C-C , Kim Ki-Wook , Taneja Reshma , Randolph Gwendalyn J , Edelson Brian T
Published: January 2023

Tissue-resident macrophages occupy key roles in immunity and physiology, both as sensors of and first responders to homeostatic perturbations. The majority of resident macrophages derive from embryonic progenitors and self-maintain locally as unique tissue-specific populations. The transcriptional basis for tissue-resident macrophage identity is poorly understood. We have observed that the transcription factor basic helix loop helix, member e40 (Bhlhe40) is highly expressed in a subset of hematopoietic cell types, including peritoneal macrophages. Based on these data, we hypothesized that Bhlhe40 is a key part of the peritoneal macrophage transcriptional network and adapts nascent macrophages to the functional demands of the peritoneal niche. We have found that peritoneal macrophages are selectively reduced in Bhlhe40−/− mice, in contrast to other resident macrophages. Furthermore, in the absence of Bhlhe40 the remaining large peritoneal macrophages (LPMs) are replaced by monocyte-derived cells to a greater extent than in wildtype mice. Bhlhe40-deficient peritoneal macrophages exhibit altered polarization and self-renewal. Mixed bone marrow chimeras and other approaches demonstrated a specific, intrinsic defect in mature Bhlhe40−/− LPMs. Using models of peritoneal immunity, we observed impaired responses of Bhlhe40-deficient peritoneal macrophages. Our preliminary findings demonstrate a role for Bhlhe40 in peritoneal macrophage biology and lead us to propose that Bhlhe40 transcriptionally controls a tissue-specific program in macrophages tailored to the peritoneal environment.
Preferential perinatal thymic generation of skin-homing CCR10+ NK1.1+ innate lymphocytes for establishment of skin homeostasis
Authors: Xu Ming , Yang Jie
Published: January 2023

Innate and innate-like lymphocytes have particularly important contribution to the establishment of local immune system in skin of newborn and infant stages, when adaptive immune cells are not fully established. However, the development pathway of the skin innate and innate-like lymphocytes is not well understood. Herein we report discovery of a novel process that generates skin-homing group 1 innate lymphoid cells (ILC1s) in fetal/neonatal thymi, which is important for regulation of commensal bacterial colonization in the skin. We found that fetal/neonatal but not adult thymus preferentially supports generation of a unique population of skin-homing CCR10+NK1.1+ ILC1s. The thymic stroma-associated Notch/ligand axis promotes the generation of CCR10+ NK1.1+ ILC1s, which is suppressed in adult thymi by developing abT cells. Infant mice that were depleted of NK1.1+ ILCs by in utero anti-NK1.1 antibody treatment had a significantly increased ratio of commensal bacteria in skin-draining lymph nodes relative to skin compared to untreated control mice. In addition, the skin of the NK1.1 antibody-treated mice had a profound alteration in composition of commensal bacteria. Our study suggests that the preferential generation of skin-homing ILCs in perinatal thymi is a developmentally programmed process to target immune cells to the skin to regulate the early colonization of commensal bacteria.
Secreted IL12p40 finds IL12p35 in the extracellular milieu to generate functional IL-12 cytokine
Authors: Gerber Allison N , Abdi Kaveh , Singh Nevil J
Published: January 2023

IL-12 is a cytokine produced by dendritic cells (DCs) that critically regulates the differentiation of naïve T cells into IFNγ producing effectors. The canonical view is that IL-12 is secreted only after the 40 kDa (p40) and 35 kDa (p35) subunits form a heterodimer inside a DC. However, the p40 subunit is also released from DCs as a monomer whose function is unclear. We previously hypothesized that secreted p40 could bind p35 in the extracellular milieu to generate functional IL-12 cytokine. Indeed, in vitro experiments using lysates of CHO cells that overexpressed p40 and p35 showed that such a heterodimer could stably form outside the cell. In order to evaluate if such an extracellular assembly happens in vivo, mixed bone marrow chimeras were set up using p35−/− and p40−/− cells. In this model, since no cell can make both p40 and p35, any IL-12 activity would have to be generated by extracellular assembly of these subunits. Infection of such chimeric mice with Leishmania major showed IFNg production in the draining lymph nodes, suggesting that functional IL-12 may be able to assemble outside of DCs in vivo.
Interleukin-4 (IL-4) maintains lymphocyte-stromal cell axis in peripheral lymph nodes
Authors: Selva Diana Cortes , Ready Andrew L , Fairfax Keke C
Published: January 2023

Interleukin-4 (IL-4) is a critical cytokine in the differentiation of Th2 cells, B-cells, and antibody isotype switching. Despite the myriad of functions known to be mediated by IL-4, its role in the spatial organization of lymph node compartments is not well understood. Our studies demonstrate that IL-4 is vital for proper positioning of B cell, T cell and CD11c+ dendritic cells in lymph nodes in steady state, and that the lack of IL-4 and IL-4 receptor (IL-4Rα) correlates with a diminished immune response to Schistosoma mansoni antigens. The absence of IL4 and IL-4R resulted in decreased germinal center (GC) formation, T follicular helper (Tfh) and plasma cells expansion in peripheral lymph nodes in response to this Th2 polarizing antigen, but not to Th1 polarizing Toxoplasma gondii antigens. Stromal cell populations are known to support lymphocyte organization and function within secondary lymphoid tissues. Importantly, we observed a marked disorganization of follicular dendritic cells (FDCs), CD31+ lymphatic, and blood endothelial cells in mice lacking IL4R, suggesting that the lymphocyte-stromal cell axis is maintained by IL-4. Our study also shows that absence of IL-4Rα correlates with significant down-regulation of Lymphotoxin-beta (LTβ) and Lymphotoxin alpha (LTα) gene expression, which is vital for the development of specific stromal populations. Altogether, our results reveal a previously unexplored role of IL-4 in the maintenance of peripheral lymphoid organ microenvironments during an immune challenge as well as in homeostasis.
Hypoxia-inducible factor-1 mediated CX3CR1 expression correlates with progression and metastasis in ovarian cancer
Authors: Singh Santosh Kumar , Lillard James W. , Singh Rajesh
Published: January 2023

Chemokines mediate the important roles in organ-specific homing of various cancer cells, including ovarian cancer (OvCa) to distant organs that may favor or inhibit cancer progression and metastasis. Hypoxia, a common phenomenon in solid tumors, is associated with a malignant cancer phenotype. Here, we provided the evidence that hypoxia up-regulate CX3CR1 expression and lead to an increased chemotactic response to CX3CL1 in OvCa cells. Hypoxia- induced genes are widely involved in metastasis-associated functions; and also undergo epithelial-to-mesenchymal transition (EMT). In the present study, we found that CX3CR1 expression was significantly increased in OvCa cells (OVCAR-3, SW626 and TOV-112D) following exposure to hypoxia. Flow cytometry, RT-PCR, western blot, Immunohistochemistry and immunofluorescence techniques were used to quantify the expression of chemokine CX3CR1and CX3CL1 in OvCa. Moreover, a significant higher expression of CX3CR1 under hypoxia compared to the normoxic condition in OvCa. Furthermore, we demonstrated that OvCa cells display a marked increase in hypoxia-induced CX3CR1-dependent PI3K/Akt/mTOR and MAPK signaling molecules as well as EMT markers and matrix metalloproteinase (MMPs) which are involved in cancer progression and metastasis of OvCa cells in hypoxic condition. These data demonstrate hypoxia pathway regulate the expression of CX3CR1 that may contribute to progression and metastasis of OvCa.
Pro-inflammatory condition-induced tumor dormancy in pre-metastatic reservoir thymus
Authors: Sizova Olga , Kuriatnikov Denis , Su Dong-Ming
Published: January 2023

Cancer patients suffering from metastatic relapse is the major cause of death. This is attributed to some undetectable minimal number of tumor cells, which are able to resist radio-chemotherapy, being in dormant state at some organs of the body. The largest T-lymphoid organ, thymus, has just recently been suggested as this kind of organ to be a pre-metastatic reservoir for tumor cell dormancy, and eventually relapse. We hypothesize that infection-, drug- and aging-resulted thymic atrophy induces inflammatory conditions to create this tumor premetastatic reservoir. Our observations in tumor-inoculated mouse model show that various cancer cells could be retained in the thymus. This retention ratio (in the thymus vs lymph nodes) was particularly high in the naturally-aging- or drug-caused atrophied thymus. We found that genotoxic chemotherapy (Doxorubicin) can lead to changes in thymic microenvironment, characterized by activation of p53 in thymic epithelial cells (TECs), induction of TEC senescence, and an increase of pro-inflammatory factors. These thymic conditions were able to confer tumor cell capacity to anti-apoptosis. We also found that dormant tumor cells in the thymus have a capacity to induce tumor recurrence in the distant organs. Our pilot experiments suggest that activation of p53 in TECs induced by DNA-damage upon Doxorubicin treatment promotes senescence, in which SASP (senescence-associated secretory phenotype) is activated, thereby leading to tumor cell dormancy/chemo-resistance. This work is clinically relevant because our findings may help to determine a new target to prevent tumor relapse after chemotherapy.
IRF8 deficiency in colonic epithelial cells promotes inflammation-mediated colon tumorigenesis and myeloid-derived suppressor cell differentiation.
Authors: Ibrahim Mohammed L. , Liu Kebin
Published: January 2023

IRF8 is a member of the nine IRF family proteins that was originally identified as a key transcription factor for myeloid cell lineage differentiation. IRF8 deficiency leads to deregulation of myelopoiesis and accumulation of CD11b+Gr1+ immature myeloid cells that phenotypically and functionally resemble tumor-induced myeloid-derived suppressor cells (MDSCs). It has since been documented that IRF8 also plays important roles in other immune cells, including B, Natural Killer, Dendritic and T cells. IRF8 expression and function has recently been extended to non-hematopoietic cells, including epithelial cancer cells and heart cells. IRF8 is often silenced by its promoter DNA methylation in cancer cells. However, low level of IRF8 is expressed in cancer cells and IRF8 is IFNg inducible in cancer cells. IRF8 acts as an apoptosis inducer. To determine the function of IRF8 in spontaneous tumor development, we generate IRF8 deficient mice with IRF8 KO only in colonic epithelial cells (termed IRF8 CKO mice). Unlike conventional IRF8-deficient mice, IRF8 CKO mice phenotypically resemble WT mice. However, IRF8 CKO exhibited greater susceptibility to AOM-DSS-induced colon cancer formation as compared to WT mice. Although no differences were observed in MDSC profiles between WT and IRF8 CKO mice, tumor-bearing IRF8 CKO mice have significantly higher level of CD11b+Gr1+MDSCs than tumor-bearing WT mice. Our data determine that IRF8 functions in colonic epithelial cells as a spontaneous colon cancer suppressor and also functions in colon carcinoma cells to mediate MDSC differentiation in tumor-bearing host in vivo.
The Nrf2/Interleukin-17D axis as a novel target for cancer immune therapy
Authors: Seelige Ruth , Saddawi-Konefka Robert , Gross Emilie T.E. , Levy Eric , Searles Stephen C. , Washington Allen , Santosa Endi K. , Liu Beichen , O’Sullivan Timothy E. , Harismendy Olivier , Bui Jack D
Published: January 2023

The oxidative stress-sensing transcription factor Nrf2 has been studied by cancer researchers as a double-edged sword that can either promote or inhibit cancer. Previous findings have shown that Nrf2 can act as a tumor suppressor by detoxifying carcinogens. Paradoxically, when cancer cells express Nrf2, they are protected from oxidative stress and certain chemotherapies, thus explaining why some cancer cells constitutively activate Nrf2 as a tumor promoter. We recently found that the cytokine Interleukin (IL)-17D mediates the recruitment of natural killer (NK) cells to tumor sites, thereby leading to tumor rejection. Here, we show that Nrf2 induced IL-17D in primary tumors and tumor cell lines. Moreover, the known Nrf2-activator tBHQ induced Nrf2 and IL-17D simultaneously, without creating harmful oxidative stress. Applying a tBHQ-containing skin cream to established tumors led to NK cell-mediated tumor regression, and this depended on IL-17D and Nrf2. We propose that the use of tBHQ or other Nrf2 activators can intiate a tumor surveillance pathway involving IL-17D and NK cells, thus mediating cancer regression and bearing potential for cancer immune therapy.
Co-localization of vimentin with a tumor associated antigen identified by the monoclonal antibody H5 in 9L gliosarcoma cells and normal glial cells from Fischer 344 rats
Authors: Davis Nancy A , Redd Joan M
Published: January 2023

Previously, we used a monoclonal antibody, designated H5, to isolate a tumor-associated antigen (TAA) expressed by 9L, a gliosarcoma line isolated from Fischer 344 (F344) rats. This TAA was tentatively identified as vimentin via mass spectrometry. In this project, we identified the location of this TAA and vimentin on 9L gliosarcoma and normal rat glial cells using confocal fluorescent microscopy. Vimentin and the TAA always co-localized which suggests that the TAA bound by H5 is a moiety on the vimentin protein. In addition, 9L expressed an increased level of vimentin compared to normal rat glial cells. Glial cells were examined from neonatal, 6 week, and adult rats. The glial cells expressed less vimentin than 9L gliosarcoma tumor cells at all ages. These results suggest that vimentin is upregulated as part of the tumor phenotype giving a possible mechanism for detecting transformed cells of this tumor type.
Targeting IL-17 for cancer-associated inflammation and immunity
Authors: Wang Kepeng
Published: January 2023

Interleukin-17A (IL-17A) is a proinflammatory cytokine whose expression has been linked to rapid malignant progression of colorectal cancer (CRC) and resistance to therapy. Ablation of IL-17A or its type A receptor (IL-17RA) in mouse models of colonic tumorigenesis confirmed their tumor-promoting function. IL-17RA signals directly within transformed colonic epithelial cells (enterocytes) to promote early tumor development. IL-17RA engagement activates ERK, p38 MAPK and NF-κB signaling and promotes the proliferation of tumorigenic enterocytes who just lost expression of the APC tumor suppressor. Although IL-17RA signaling also controls production of IL-6, this mechanism makes only a partial contribution to colonic tumorigenesis. Combined treatment with chemotherapy, which induces IL-17A expression, and an IL-17A neutralizing antibody enhanced the therapeutic responsiveness of established colon tumors. Consistent with the notion that IL-17 promotes tumor-associated inflammation and inhibits anti-tumor immunity, ablation of IL-17RA resulted in reduced infiltration of CD11b+ cells and elevated numbers and activity of cytotoxic T lymphocytes in colorectal tumors. Using anti-IL-17 as an adjuvant therapy may improve the efficacy and safety of current cancer immunotherapeutic schemes.
Dual role of SIRPα in macrophage activation: inhibiting M1 while promoting M2 polarization via selectively activating SHP-1 and SHP-2 signal
Authors: Shi Lei , Bian Zhen , Liu Yuan
Published: January 2023

Macrophages polarization into proinflammatory M1 or alternative M2 states, which plays a central role in inflammation and host defense, is tightly controlled by multiple regulatory molecules. Here we examined the role of signal regulatory protein α (SIRPα), which negatively regulates leukocyte responses, in regulating macrophage polarization and function. Compared to WT macrophages, macrophage obtained from SIRPα deficient (SIRPα−/−) mice demonstrated an enhanced response to M1 induction but were refractory to M2 induction, as indicated by the increased expression of M1-related markers CD86, MHC-II, iNOS, IL-1β, IL-12 and TNF-α under M1-skewed LPS/IFNγ treatment but the defective expression of anti-inflammatory mediators CD206, Arg1, IL-10 and TGFβ during IL-4 induced M2 activation. Furthermore, ligating SIRPα by CD47 extracellular domain (mCD47-AP) suppressed M1 polarization but enhanced M2 polarization in WT macrophages, whereas had no effect on SIRPα−/− macrophage polarization. Interestingly, under M1 and M2 activation, macrophage SIRPα was found to be tyrosine-phosphorylated and selectively associated with SHP-1 and SHP-2, respectively. Mechanistic studies indicated that SIRPα deficiency promoted M1 but attenuated M2 polarization of macrophages by enhancing the PI3k/Akt2 signaling pathway. Overall, our findings demonstrated for the first time that SIRPα plays an important role in modulating macrophage polarization, inhibiting M1 while promoting M2 phenotypic polarization through selectively activating SHP-1 and SHP-2 signaling.
The novel IgG-receptor FCRL5 is able to distinguish IgG2 disulfide isomers and displays increased affinity for heat-stressed IgG2
Authors: Alabi Oyeleye A , Dement-Brown Jessica , Tolnay Mate
Published: January 2023

Human Fc receptor-like 5 (FCRL5) is a novel IgG receptor that senses the whole IgG molecule, including the Fc and F(ab′)2 portions. Using surface plasmon resonance (Biacore) technology, we established that FCRL5 binds human IgG via a complex mechanism, where IgG isotype is just one determinant of the interaction. In particular, individual IgG2 samples were found to display a thousand-fold range of affinities as well as distinct kinetics, indicating that additional factors beyond the isotype affect binding. We further investigated two potential factors that may affect the interaction of IgG2 with FCRL5; structural heterogeneity related to IgG2 disulfide isoforms and charge variants mostly due to deamidation. We generated IgG2 samples enriched for either of the disulfide isoforms, IgG2-A and IgG2-B, and assessed their binding to FCRL5 using Biacore. We found that the IgG2-A isoform bound FCRL5 with up to 80-fold higher affinity than the IgG2-B isoform. IgG2 samples were also kept at 40°C for up to four weeks, a condition known to promote deamidation. We found that FCRL5 displayed higher affinity for heat-stressed IgG2, suggesting that the interaction is sensitive to deamidation of the IgG2. We conclude that FCRL5 is able to discriminate two independent changes in the IgG2 molecule, the disulfide isoform structure as well as charge variants likely related to deamidation. Of note, both conversion of disulfide isoforms and deamidation of the molecule are known to occur in vivo, on time scales comparable to the half-life of the IgG2 molecule. Therefore, our studies raise the possibility that FCRL5 discerns IgG2 variants produced in vivo in days or weeks, providing insight into the nature of the physiological FCRL5 ligand.
Discovery of a novel transcriptional regulator of sustained TLR-mediated inflammatory responses
Authors: LIN BIN , Dutta Bhaskar , Oh Kyu-Seon , Fraser Iain D.C.
Published: January 2023

Toll-like receptor (TLR) pathways have been intensively investigated for almost two decades. However, since most studies have focused on the initial signal transduction and transcription events, the mechanisms of late-sustained gene expression are poorly understood. To address this issue, we conducted an RNAi screen for regulators of TLR-induced IL-6 secretion in RAW264.7 macrophages, and identified a novel putative regulator of this process (Hit4). Hit4 was specifically required to sustain the late expression of genes including Il6, Il1a, Nos2 and Lcn2, but did not affect induction of transiently expressed genes such as Nfkbiz, Egr1, and Egr2. This effect of Hit4 was confirmed by CRISPR/Cas9 gene editing and by RNAi knockdown in primary macrophages. Most interestingly, Hit4 functioned specifically in sustaining rather than initiating transcription, as Hit4 depletion showed no effect on the early mRNA transcription before 2 hr for all gene classes. Further mechanistic investigation revealed that Hit4 functioned at the transcriptional level rather than post-transcriptional level, as Hit4 enhanced the level of nascent transcripts at later time points. Moreover, Hit4 KO RAW264.7 macrophages failed to sustain the response to persistent LPS stimulation, showing a response similar to that observed for transient LPS stimulation. This suggests a role for Hit4 in discriminating brief spurious pulses of infectious signal from a more sustained real infection signal. In summary, we have identified a novel transcriptional regulator that functions to sustain transcription of late inflammatory genes, providing important new insight to the control of TLR-induced gene program.(Supported by the Intramural Research Program of NIAID, NIH)
Characterization of the role of NLRP10 in dendritic cells
Authors: Vacca Maurizio , Mortellaro Alessandra
Published: January 2023

The ability of the immune system to recognize a large variety of microbial-derived molecules relies on diverse families of pattern-recognition receptors (PRRs). One important family of PRRs is the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). Among all the NLRs, NLRP10 has unique features, as it is the only member lacking the ligand-binding leucine-rich-repeat (LRR) domain. NLRP10 protein is poorly characterized and its functions are still unknown. Nlrp10 is expressed almost in immune and non-immune tissues. In this study, we characterized the role of the murine Nlrp10 in host immune response. We firstly assessed the role of Nlrp10 in DCs by examining Nlrp10−/− DC ability to present protein antigens and thereby activate T cells. We found that ovalbumin-specific CD4+ T cells when co-cultured with Nlrp10−/− DCs produce less IFNγ compared to T cells co-cultured with WT DCs. We then measured the expression of some co-stimulatory molecules (CD80, CD86), as well as MHC class I and II in immature and LPS-matured Nlrp10−/− DCs. Expression levels of these molecules are similar in WT and Nlrp10−/− DCs. Furthermore, we evaluated the release of specific cytokines responsible for T cell activation in DC/T cell co-cultures. Preliminary data suggest that Nlrp10−/− DCs have a defect in IL-12 release as a result of impaired NFkB signaling. Nlrp10 contribution in the pathways regulating IL-12 production is currently under investigation. Since associations between NLRP10 and inflammatory disorders are emerging, a better understanding of the underlying mechanisms regulated by NLRP10 in immune response may help us to identify potential new targets for anti-inflammatory therapies.
Post-transcriptional elements within NLRP3 mRNA set the activation threshold of the inflammasome.
Authors: Varela Stephanie Nichole , Schwerk Johannes , Forero Adriana , Hong MeeAe , Arroyo Nicole , Chou Bryan , Savan Ram
Published: January 2023

NOD-like receptor protein 3 (NLRP3) is an intracellular sensor and a major component of the canonical inflammasome pathway, which causes inflammation and cell death when activated by two temporally discrete signals. While novel putative activators of the NLRP3 inflammasome have been identified, the mechanisms that regulate expression of NLRP3 itself are less understood. Alternative polyadenylation (AP) is a mechanism by which cells modulate expression through varying the length of the 3′ untranslated region (3′UTR). Through AP, the longer forms may provide unique regulatory elements, that allow for differential regulation of mRNA stability and translation. Here we sought to thoroughly characterize the features of the NLRP3 3′UTR and hypothesized that AP may significantly control the threshold of inflammasome activation. We examined AP in THP-1 monocytes and found that they express both long and short forms, with the long form demonstrating decreased stability relative to the short. We have attributed these differences to the presence of AU-rich elements (ARE), as ablation of AREs results in increased stability of the long NLRP3 mRNA. Additionally, we have demonstrated that a known post-transcriptional regulator of NLRP3, miR-223, preferentially regulates the long transcript as well as augments the kinetics of pyroptotic cell death. These studies demonstrate that alternative polyadenylation of the 3′UTR of NLRP3 has a marked impact on the threshold of inflammasome activation.
Inflammatory ILC2: An IL-25-activated circulating ILC population with a protective role during helminthic infection
Authors: Huang Yuefeng , Mao Kairui , Chen Xi , Kawabe Takeshi , Li Weizhe , Zhu Jinfang (Jeff) , Urban Joseph F. , Germain Ronald N , Paul William E
Published: January 2023

Innate lymphoid cells (ILCs) play important roles in host defense, tissue repair, metabolic homeostasis and inflammatory diseases. While ILCs are generally considered to mediate these functions in a tissue-resident manner, whether ILCs can move between sites when infection demands has not been carefully explored. We have reported the existence of an ILC population, inflammatory ILC2 (iILC2) cells, which are not present in the steady state but appear in high numbers in lung, liver, mesenteric lymph nodes and spleen upon IL-25 or Nippostongylus brasiliensis infection. iILC2 cells develop into natural ILC2-like cells and contribute to the expulsion of N. brasiliensis worms. They can also acquire IL-17-producing ability and provide protection against Candida albicans, indicating their multipotentiality or plasticity. Our recent data reveals that iILC2 cells are circulating cells, exchanging to a much greater extent between parabiotic mice than other ILC populations. iILC2 cells that appear after IL-25 administration or helminthic infection in peripheral, non-gut tissues such as the lung arise from distant resting ILC2 cells residing in the intestinal lamina propria. IL-25 induces rapid proliferation of these intestinal ILC2 cells and a change in their sensitivity to S1P-mediated chemotaxis, leading to lymphatic entry, blood circulation, and accumulation in many non-gut sites. These gut-derived cells are critical for anti-helminth immunity and tissue repair in the bowel and lung. Thus, inflammatory ILC2 cells are a circulating ILC population that is derived from the intestine but plays important roles in peripheral tissue host defense.

This work was supported by the Intramural Research Program of NIAID, NIH and by the USDA.
Analysis of caspase-1 regulated transcriptome in various tissues lead to identification of novel IL-1β, IL-18 and sirtuin-1 intendent pathways
Authors: Nanayakkara Gayani K , Li Ya-feng , Yu Sun , Choi Eric T , Wang Hong , Yang Xiao-Feng
Published: January 2023

Caspase-1 is prominently involved in bridging disease risk factors/ danger signals to inflammatory response via its downstream targets such as IL (interleukin)-1β, IL-18, and Sirt-1 (Sirtuin-1). The microarray datasets derived from caspase-1 knockout tissues indicated that caspase-1 function can significantly impact the transcriptome. However, it is not known whether all effects exerted by caspase-1 on transcriptome are mediated only by its well-known substrates. Therefore, we hypothesized that the effects of caspase-1 on transcriptome may be partially independent from IL-1β, IL-18, and Sirt-1. To determine new global and tissue-specific gene regulatory effects of caspase-1, we took novel microarray data analysis approaches including Venn analysis, cooperation analysis, and meta-analysis. We made the following important findings: (1) Caspase-1 exerts its regulatory effects on majority of genes in a tissue-specific manner; (2) Caspase-1 regulatory genes partially cooperates with genes regulated by Sirt-1 during organ injury and inflammation in adipose tissue but not in liver; (3) Caspase-1 cooperates with IL-1β in regulating less than half of the genes involved in organismal injury, and cancer in mouse liver; (4) The meta-analysis identified 40 caspase-1 globally regulated genes across tissues, suggesting that caspase-1 globally regulates many novel pathways; and (5) The meta-analysis identified new cooperatively and non-cooperatively regulated genes in caspase-1, IL-1β, IL-18, and Sirt-1 pathways. Our findings suggest that caspase-1 regulates many new signaling pathways potentially via its known substrates and also via transcription factors and other proteins that are yet to be identified.
Assessment of the dual mechanism of inflammatory inhibition by the novel JODI series of compounds
Authors: Szollosi Doreen , Manzoor Mohammed , Lucero Victoria , Dhuguru Jyothi , Edafiogho Ivan , Ghoneim Ola
Published: January 2023

Previous experiments in our laboratory have screened several novel compounds for anti-inflammatory activity, including enaminone E121 and the related JODI series. These studies have concluded that these compounds decrease the release of pro-inflammatory cytokines TNF-α and IL-6 after stimulation with bacterial lipopolysaccharide (LPS). Additionally, the JODI compounds, which have an N-arylpiperazine ring added to the enaminone pharmacophore appear to also have chemokine inhibition activity. These studies in our laboratory suggest that the JODI compounds not only have a drug target in the Toll-like receptor pathway (after stimulation with LPS) but also a drug target in the MCP-1/CCR2 pathway. Ongoing studies are aimed at evaluating these compounds for their specific drug targets by assessing kinases including p38 which appears to be decreased after JODI treatment, as well as NF-kB translocation. Taken together, the JODI compounds represent a potentially novel series of anti-inflammatory compounds for inflammatory disorders in which efficacious treatments are urgently needed.
The nuclear receptor REV-ERBα modulates Th17 cell-mediated autoimmune disease
Authors: Loo Chin San , Chang Christina , Zheng Ye
Published: January 2023

T helper 17 cells (Th17) play an important role in controlling extracellular bacteria and yeast infections; however, dysregulated Th17 response can lead to detrimental autoimmune diseases such as multiple sclerosis. Th17 cell differentiation relies on IL6/TGF-β-induced expression of retinoic acid receptor-related orphan nuclear receptor RORγt, which is able to induce Th17 effector cytokines such as IL17A. Therefore, antagonizing RORγt activity may prove to be one of the therapeutic strategies for ameliorating Th17-mediated inflammation. Here we identify REV-ERBα (encoded by Nr1d1), a member of the nuclear hormone receptor family, as a transcriptional repressor that antagonizes RORγt-induced Il17a expression. Both RORγt and REV-ERBα bind to the same ROR response element (RORE) in Il17a promoter region. Elevated expression of REV-ERBα prevents RORγt binding to RORE and reduces Il17a expression. Furthermore, treatment with a synthetic REV-ERBα agonist, SR9009, significantly delays the onset and impedes the progression of experimental autoimmune encephalomyelitis (EAE), a Th17-mediated autoimmune disease. These results suggest that modulating REV-ERBα activity holds therapeutic potential for treatment of Th17 cell-mediated autoimmune diseases.
Characterization of varicella zoster virus-specific monoclonal antibodies
Authors: Sullivan Nicole L , Monslow Morgan , Sei Janet , Durr Eberhard , Chang Cathy , Mccausland Megan , Rouphael Nadine , Pulendran Bali , Ahmed Rafi , Vora Kalpit A
Published: January 2023

Herpes zoster (HZ or shingles) is the clinical manifestation of varicella zoster virus (VZV) reactivation. HZ typically develops as people age due to decreased cell mediated immunity. However, the relative importance of humoral immunity against VZV remains somewhat controversial. The goal of this study was to examine the breadth and functionality of VZV-specific antibodies. Direct enumeration of VZV-specific antibody secreting cells (ASC) via ELISPOT showed that Zostavax® vaccination can induce both IgG and IgA ASCs seven days after vaccination, but not IgM ASCs (IgG>IgA≠IgM). The frequency of VZV-specific ASCs was from 33–55% of the total IgG ASCs. ASCs were also single-cell sorted from five subjects (> 60 years old) and 25 VZV-specific monoclonal antibodies (mAbs) were successfully cloned and characterized. These mAbs had on average ~ 20 somatic hypermutations per VH gene, similar to the number of mutations seen in mAbs characterized after seasonal influenza vaccination (Li GM, PNAS, 2012). Fifteen of the 25 mAbs were gE-specific, whereas the remaining mAbs were gB, gH or gI-specific. The antibodies were then tested for in vitro neutralization and inhibition of cell-to-cell spread, which will be discussed. These data indicate that Zostavax® vaccination can induce a memory B cell recall response and gE is the predominant antibody target. However, the relative importance of these antibodies for protective immunity against VZV reactivation will be discussed.
Vaccine display on synthetic bacterial spores enhances protective efficacy against Staphylococcus aureus infection
Authors: Karauzum Hatice , Updegrove Taylor , Wu I-Lin , Datta Sandip K , Ramamurthi Kumaran
Published: January 2023

Background

Modification of bacterial spore surfaces with proteins has previously been proposed as vaccine display platforms, however, relied on viable spores.

Objective

In the present study we describe how synthetic spore husk-encased lipid bilayers (SSHELs) can be utilized as a display platform to present protein antigens like ovalbumin and mutant staphylococcal alpha toxin (HlaH35L) as vaccines. This platform is based on 2 self-assembling proteins SpoVM and SpoIVA from Bacillus subtilis.

Methods

C57BL/6 mice were immunized with SSHEL (with or without adjuvant), antigen or antigen covalently bound to SSHEL (SSHEL_antigen) thrice intramuscularly at two-week intervals and serum antibody titers against SpoIVA or antigens were determined 7 days after each immunization. In select experiments mice were challenged intravenously with a lethal dose of S. aureus USA300 2 weeks after last vaccination. Untreated mice served as controls.

Results

Immunization of mice with SSHEL induced a similar antibody response towards SpoIVA as SSHEL that was adjuvanted in CpG or MPL-A suggesting adjuvant properties of this synthetic lipid-bilayer construct. Post vaccination serum of mice immunized with ovalbumin covalently bound to SSHEL (SSHEL_OVA) or HlaH35L covalently bound to SSHEL (SSHEL_HlaH35L) contained SpoIVA specific as well as antigen-specific (OVA or HlaH35L) antibodies. Infection of mice with a lethal dose of USA300 resulted in 100% survival of mice immunized with SSHEL_HlaH35L compared to 20% survival of non-immunized mice (P=0.01), and 40% survival of mice immunized with HlaH35L (P=0.049).

In progress

In ongoing studies we are evaluating the mechanism of action of SSHEL_HlaH35L that lead to protection during infection.
miR-148a controls plasma cell differentiation and survival
Authors: Pracht Katharina , Meinzinger Julia , Daum Patrick , Côrte-Real Joana , Hauke Manuela , Schulz Sebastian , Roth Edith , Wittmann Jürgen , Jäck Hans-Martin
Published: January 2023

microRNAs (miRNAs) are critical regulators of central and Ag-driven B cell development. However, it is unknown whether miRNAs regulate the differentiation of B cells into plasma cells (PCs) and control the survival of long-lived PCs.

We recently showed that miR-148a, the most abundantly expressed miRNA in PCs, is upregulated in activated B cells and promotes in vitro plasmablast differentiation and survival by targeting Bach2, MiTF, PTEN and Bim.

To investigate if miR-148a supports PC differentiation in vivo, we established a conditional B cell-specific miR-148a knock-out mouse. Non-immunized mice showed a significant decrease in serum Ig, antibody-secreting cells (ASC) and splenic PCs. 28d after boost, TNP-KLH-immunized 148a-KO mice had lower serum TNP-IgG and decreased numbers of splenic ASCs and bone marrow (BM) PCs.

To ask whether miR-148a controls PC survival, we generated tamoxifen-inducible miR-148a deficient mice. Tamoxifen-mediated miR-148a deletion, 28 days after boost with TNP-KLH, resulted in reduced numbers of plasmablasts and IgG-ASCs in the spleen and of resting PCs and IgG-ASCs in BM. In parallel, increased numbers of resting PCs were found in the blood, indicating a possible egress from survival niches.

These findings support the hypothesis that miR-148a controls the differentiation of B cells into plasmablasts as well as the survival of long-lived PCs. We will now identify the underlying mechanism by a biosystems approach utilizing transcriptome and proteome profiles of fresh follicular B cells and BM PCs.

Proteome analysis was done in collaboration with the Warscheid lab at the University of Freiburg.
Affinity maturation of anti-lipid IgG antibodies produced via germinal center
Authors: Baeza Carlos Wong , Resendiz Albany , Landa Carla , Angeles Oscar , Tescucano Alonso , Wong Carlos , Flores-Romo Leopoldo , Baeza Isabel
Published: January 2023

Non-bilayer phospholipid arrangements (NPA) are lipid associations different to the bilayer and are found on cell membranes; they participate in different cellular functions and are transient, but when they are stabilized by drugs such as chlorpromazine, they become immunogenic and induce the formation of anti-NPA antibodies. Mice that receive stabilized NPA share several characteristics with patients with systemic lupus erythematosus, such as the presence of anti-cardiolipin, anti-histone and anti-nuclear antibodies, lupus band, malar exanthema and glomerulonephritis. Mice with this lupus model have been shown to develop the disease as a consequence of the production of anti-NPA antibodies; in addition, most of these antibodies are of the IgG class and are produced mainly by the germinal center pathway. In the case of protein antigens, germinal centers are the sites where class-switch recombination, somatic mutation, memory generation and affinity maturation occur. To determine if affinity maturation, a hallmark of the antibodies produced via germinal center reactions, occurs in the anti-NPA IgG antibodies produced in germinal centers, we performed antigen-specific ELISAs, both in the absence and in the presence of urea. We observed that the percentage of urea-resistant (high affinity) anti-NPA IgG antibodies in relation to the total anti-NPA IgG antibodies increases over time, and it correlates with the number of NPA-specific germinal center B cells.