ImmunoHorizons最新文献

The immune regulatory gene PTPN22 is expressed in all immune cells and encodes lymphoid protein (Lyp) in humans and the ortholog PEST domain-enriched phosphatase (PEP) in mice. The PTPN22 alternative allele, 1858C>T, is expressed in 5% to 15% of the North American population and is strongly associated with the development of autoimmune disease while simultaneously capable of providing protection during viral infection and cancer. In murine models, significant progress has been made in elucidating the molecular mechanisms by which PEP and its pro-autoimmune variant (PEP-R619W) modulate T-cell function, yet their influence on non-T-cell pathways, such as antigen-presenting cell cytokine production, remains less defined. Previously, it was reported that PEP promotes type I interferon (IFN-I) production in dendritic cells (DCs) and macrophages following TLR4 stimulus. Here, we show that contrary to previous results, PEP and the PEP-R619W variant do not mediate IFN-I production in DCs and macrophages following exposure to LPS, 3p-hpRNA, or coronavirus mouse hepatitis virus strain A59. We attribute the prior findings to mouse strain-specific differences and conclude that factors independent of PEP may be regulating IFN-I production in these studies. We further show that PEP and its R619W variant distinctly modulate the production of TNF-α, IL-12, and IL-2 in DCs following LPS stimulus. Taken together, our results challenge the current understanding of the role of PEP during inflammation while providing new insight into how the PEP-R619W variant may alter myeloid cell function during disease.

Osteopontin is a protein with many physiological roles widely expressed by many cell types, tissues, and bodily fluids, including breastmilk. The functions of breastmilk osteopontin are not clearly defined, however, it is known to impact intestinal and brain development in infants. Although it has been shown that endogenous osteopontin influences the survival of intestinal intraepithelial lymphocytes (IEL), the impact of milk osteopontin on developing intestinal immune cells remains unclear. In this report, mouse models lacking expression of osteopontin were used to demonstrate that milk-derived osteopontin is important for the development of IELs, with observed effects in both juvenile and adult mice. These changes are most prevalent in IELs expressing CD8αα: however, the impact of these alterations is unclear, as mice with disrupted IEL compartments are not more susceptible to intestinal inflammation induced by DSS or Citrobacter rodentium infection.

Cas9-expression from the Rosa26 "safe harbor" locus are widely used for gene manipulation and Crispr-based screening. Recently, experimental evidence suggested that macrophages isolated from Rosa26-Cas9 mice may have signaling differences compared to control mice in terms of TRIF signaling downstream of TLR3 and TLR4. As we frequently use the Rosa26-Cas9 mice made by Feng Zhang (Cas9-FZ, Jackson Laboratory stock No. 026179), arguably the strain with the widest distribution and utilization, we were motivated to test macrophage signaling in these mice under our conventional conditions. We used different macrophage polarization and signaling conditions combined with RNA sequencing and measurement of TLR signaling by immunoblotting. Our results suggest that the Cas9-FZ mice bear no obvious defects in any commonly used macrophage signaling pathway. We document the differences in our macrophage culture techniques compared to Raychowdhury et al., which may aid in how individual laboratories use Cas9-expressing macrophages, especially for focused or genome-wide screening.

The need for effective infectious disease vaccines has become an inescapable topic in recent years. Continued development of next-generation vaccines that provide robust protective immunity is imperative. Such vaccines will likely include adjuvants that avoid excessive adverse reactions while allowing for dose and antigen sparing. Bacterially derived TLR4 agonist, BECC438, has recently emerged as a lead adjuvant candidate across several experimental models of infectious disease, including Yersinia pestis (plague), human papillomavirus, influenza A (flu), SARS-CoV-2 (COVID-19), and Shigella spp (gastrointestinal infection). To confirm that BECC438 is a high-quality immunoadjuvant, even without antigen from an infectious pathogen, studies presented here use the model antigen ovalbumin in a murine prime-boost vaccine model. Durable and more balanced production of antibody isotypes IgG1 and IgG2 is observed when the bacterial enzyme combinatorial chemistry adjuvant is used, as compared with the classic adjuvants aluminum salts (Alhydrogel) and synthetic monophosphorylated lipid A-PHAD (phosphorylated hexaacyl disaccharide). Antibody responses are maintained for at least 18 wk postvaccination. Observed immune metrics maintained similar trends across males, females, and genetic backgrounds, including C57BL/6, BALB/c, and CD-1 (outbred) mice, with males overall showing a lower production of IgG2c. In vitro analysis of C57BL/6 serum showed an increased half-life of ovalbumin-specific antibodies in BECC438 adjuvanted animals, indicative of a higher antigen binding affinity. These studies provide continued evidence to support the development of the BECC438 adjuvant in vaccines for human use.

Cystic fibrosis (CF) is caused by homozygous mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in multi-organ dysfunction and decreased lifespan and quality of life. A durable cure for CF will likely require a gene therapy approach to correct CFTR. Rapid advancements in genome editing technologies, including CRISPR/Cas9, have already resulted in Food and Drug Administration (FDA) approval for cell-based gene editing therapies, providing new therapeutic avenues for many rare diseases. However, immune responses to gene therapy delivery vectors and editing tools remain a challenge, especially for strategies targeting complex in vivo tissues such as the lung. Previous findings in non-CF healthy individuals reported pre-existing antibody and T cell responses to recombinant Cas9 proteins, suggesting potential additional obstacles for incorporation of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technologies in gene therapies. To determine whether pre-existing immunity to Cas9 from S. aureus or S. pyogenes was present or augmented in people with CF, anti-Cas9 IgG levels and Cas9-specific T cell responses were determined from peripheral blood samples of people with CF and non-CF healthy controls. Overall, non-CF control and CF samples displayed evidence of pre-existing antibody and T cell responses to both S. aureus and S. pyogenes Cas9, although there were no significant differences between these populations. However, we observed global changes in CF activation of Th1 and CD8 T cell responses as measured by interferon γ (IFN-γ) and tumor necrosis factor (TNF) that warrant further investigation and mechanistic understanding as this finding has implications not only for CRISPR/Cas9 gene therapy for people with CF but also for protection against infectious disease.

Fibroblasts are structural cells primarily involved in tissue remodeling, but recent single-cell RNA sequencing (RNA-seq) transcriptomic data have highlighted their potential to display molecules linked to inflammation. The factors that drive such inflammatory transcriptional signatures found in patients are not clear. LIGHT (TNFSF14) is a cytokine that we previously suggested may be central to lung diseases exhibiting fibrosis and inflammation, including asthma and interstitial lung disease. With bulk RNA-seq, we then investigated the transcriptional activity of LIGHT in human pulmonary fibroblasts compared with interleukin (IL)-13 and IL-17, two other cytokines linked to lung disease. While all 3 cytokines individually induced unique and overlapping gene transcripts, when fibroblasts were stimulated with LIGHT and IL-13 they upregulated more inflammatory transcripts including CCL2, CCL26, CXCL2, CXCL3, CXCL5, CXCL6, IL32, CSF2, VCAM1, ICAM1, IL18R1, IL1RL1, TNFRSF12A, TNFRSF4, TNFRSF8, ITGA2, ITGA4, and ITGAV, and when stimulated with LIGHT and IL-17, inflammatory transcripts included CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL32, IL33, CSF2, TSLP, IL1A, IL6, IL18, VCAM1, ICAM1, IL18R1, IL1RL1, TNFSF4, TNFRSF4, TNFRSF8, ITGA2, ITGA4, and ITGAV. Furthermore, multiple cell cycle-related transcripts were induced with these combinations. Providing potential disease significance, portions of the fibroblast transcriptional signatures induced in vitro were found to be present in subsets of fibroblasts defined by single-cell RNA-seq isolated from patients with interstitial lung disease. This study therefore highlights the synergistic activities of LIGHT with other classical cytokines to regulate transcription in pulmonary fibroblasts and infers the involvement of LIGHT in shaping fibroblast phenotypes observed in chronic lung disease.

Hypermobile Ehlers-Danlos Syndrome (hEDS) is a poorly understood connective tissue disorder that lacks molecular diagnostic markers. This study aimed to identify proteomic signatures associated with hEDS to define underlying pathophysiology and to inform objective diagnostic strategies with therapeutic potential. An unbiased mass spectrometry-based proteomic analysis of serum from female hEDS patients (n = 29) and matched controls (n = 29) was conducted. Differentially abundant proteins were analyzed through pathway enrichment and gene ontology pipelines. Prioritized candidate biomarker proteins were verified in expanded patient and control cohorts via ELISA. Cytokine array profiling was conducted to assess immune signaling patterns. Proteomic analysis revealed 35 differentially expressed proteins in hEDS, with 43% involved in the complement cascade and 80% linked to immune, coagulation, or inflammatory pathways. Pathway analyses confirmed enrichment in complement activation, coagulation, and stress responses. ELISA validation showed significant reductions in C1QA, C3, C8A, C8B, and C9 in hEDS patients, consistent across age and sex. Cytokine profiling revealed alterations in nodal immune cell mediators in hEDS patients, supporting a model of dysregulated inflammatory response. Our findings indicate a systemic immune dysregulation, particularly involving the complement system and profibrotic cytokines, as a common feature in hEDS pathophysiology. These findings challenge the traditional view of hEDS as solely a connective tissue disorder and support a revised paradigm that includes innate immune dysfunction. This immune involvement may contribute to disease pathophysiology and inform the development of biologically based diagnostic tools, enabling earlier diagnosis and guiding future therapeutic strategies.

Immunosuppressive elements within the tumor microenvironment include both regulatory T cells (Tregs) and M2 macrophages. A well-described mechanism of Treg recruitment occurs via the chemokine CCL22, and CCL22 has also recently been implicated in the polarization of tumor-associated macrophages to the M2a subtype. Our lab and others have shown that CCL22 is upregulated in cancer cells following activation of the stimulator of interferon genes (STING). STING triggers immune responses against pathogenic nucleic acids as well as self-DNA mislocalized to the cytoplasm, which can accumulate in cancer cells due to chromosomal instability, damaged mitochondria, and increased expression of LINE-1 retrotransposons. STING activation has been associated with both anti-tumor and pro-tumor immune responses, and a potential mechanism of STING-mediated immune evasion is through CCL22 upregulation. CCL22 was first characterized in macrophages, and here we investigate the effects of STING activation on CCL22 expression in macrophages and monocytes. We report that human macrophages and monocytes are resistant to CCL22 upregulation by STING, but that STING-activated macrophages and monocytes release unidentified paracrine factor(s) that dramatically increase CCL22 upregulation in cancer cells in a manner that remains STING-dependent, as evidenced by the inability of STING knockout cells to upregulate CCL22 in response to these factors. We further found that exogenous type I interferons, a major downstream product of STING activation, also upregulate CCL22 in cancer cells via a STING-dependent mechanism and that exogenous IFN-β can directly activate STING.

(SARS-CoV-2) has infected a large fraction of the human population. Currently, most individuals have developed immunity either through vaccination or natural infection. Despite this, SARS-CoV-2 booster immunizations are still recommended to reduce the risk of reinfections, but there is still limited understanding of how different booster vaccine platforms influence antibody responses. We conducted immunological studies in mice to evaluate the boosting effects of different vaccine platforms on antibody responses. C57BL/6 mice were first primed with an adenovirus serotype 5 (Ad5) vector vaccine expressing the SARS-CoV-2 spike protein. The mice were then boosted with the same Ad5-based vaccine (homologous boosting) or with a protein-based vaccine (heterologous boosting). Interestingly, the heterologous regimen (Ad5 prime/Protein boost) elicited higher binding antibody responses, relative to the homologous regimen (Ad5 prime/Ad5 boost). Similar potentiation of antibody titers was reported when mice were primed with poxvirus or rhabdovirus vectors and then boosted with protein. These findings highlight a potential advantage of protein booster immunizations to potentiate humoral immunity.

The inflammation resolution receptor lipoxin A4/formyl peptide receptor 2 (ALX/FPR2) plays a critical role in immune regulation by binding select oxylipins derived from n-6 and n-3 polyunsaturated fatty acids (PUFAs). While ALX/FPR2 is implicated in controlling inflammation initiation and resolution, its specific role in pulmonary inflammatory responses remains unclear. In this study, we investigated how genetic deletion of Alx/Fpr2 controls oxylipin levels, immune cell populations, and inflammatory cytokines under conditions of homeostasis and injury. Alx/Fpr2 knockout (KO) mice exhibited normal food intake and weight gain but showed impaired glucose and lipid metabolism. Targeted lipidomic analyses by liquid chromatography-tandem mass spectrometry revealed elevated pulmonary concentrations of n-6 and n-3 PUFA-derived oxylipins in KO mice compared to controls. Flow cytometry further demonstrated increased lung infiltration of NK cells, monocytes, and lymphoid cells, indicating a proinflammatory state in the absence of injury. Following 24 h of LPS-induced acute lung injury, IL-1β levels were elevated in KO mice, but pulmonary histopathology, immune cell numbers, and oxylipin levels were comparable to those of controls. These results suggested a protective role of ALX/FPR2 upon acute lung injury, which led us to further investigate the role of ALX/FPR2 upon 72 h of lung injury. Indeed, Alx/Fpr2 KO mice showed reduced bronchoalveolar lavage protein concentration and lower levels of IL-6 and TNF-α. Collectively, these findings demonstrate that ALX/FPR2 deficiency promotes basal pulmonary inflammation but protects against prolonged injury-induced inflammation, highlighting the context-dependent role of this receptor in pulmonary inflammation.