skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • Suppressors of Cytokine Signaling (SOCS)1 and SOCS3 Proteins Are Mediators of Interleukin-10 Modulation of Inflammatory Responses Induced by Chlamydia muridarum and Its Major Outer Membrane Protein (MOMP) in Mouse J774 Macrophages
Title: Suppressors of Cytokine Signaling (SOCS)1 and SOCS3 Proteins Are Mediators of Interleukin-10 Modulation of Inflammatory Responses Induced by Chlamydia muridarum and Its Major Outer Membrane Protein (MOMP) in Mouse J774 Macrophages
The immunopathology of chlamydial diseases is exacerbated by a broad-spectrum of inflammatory mediators, which we reported are inhibited by IL-10 in macrophages. However, the chlamydial protein moiety that induces the inflammatory mediators and the mechanisms by which IL-10 inhibits them are unknown. We hypothesized that Chlamydia major outer membrane protein (MOMP) mediates its disease pathogenesis, and the suppressor of cytokine signaling (SOCS)1 and SOCS3 proteins are mediators of the IL-10 inhibitory actions. Our hypothesis was tested by exposing mouse J774 macrophages to chlamydial stimulants (live Chlamydia muridarum and MOMP) with and without IL-10. MOMP significantly induced several inflammatory mediators (IL-6, IL-12p40, CCL5, CXCL10), which were dose-dependently inhibited by IL-10. Chlamydial stimulants induced the mRNA gene transcripts and protein expression of SOCS1 and SOCS3, with more SOCS3 expression. Notably, IL-10 reciprocally regulated their expression by reducing SOCS1 and increasing SOCS3. Specific inhibitions of MAPK pathways revealed that p38, JNK, and MEK1/2 are required for inducing inflammatory mediators as well as SOCS1 and SOCS3. Chlamydial stimulants triggered an M1 pro-inflammatory phenotype evidently by an enhanced nos2 (M1 marker) expression, which was skewed by IL-10 towards a more M2 anti-inflammatory phenotype by the increased expression of mrc1 and arg1 (M2 markers) and the reduced SOCS1/SOCS3 ratios. Neutralization of endogenously produced IL-10 augmented the secretion of inflammatory mediators, reduced SOCS3 expression, and skewed the chlamydial M1 to an M2 phenotype. Inhibition of proteasome degradation increased TNF but decreased IL-10, CCL5, and CXCL10 secretion by suppressing SOCS1 and SOCS3 expressions and dysregulating their STAT1 and STAT3 transcription factors. Our data show that SOCS1 and SOCS3 are regulators of IL-10 inhibitory actions, and underscore SOCS proteins as therapeutic targets for IL-10 control of inflammation for Chlamydia and other bacterial inflammatory diseases.  more » « less
Award ID(s):
1646729
PAR ID:
10417367
Author(s) / Creator(s):
; ; ; ;
Date Published:
Journal Name:
Mediators of Inflammation
Volume:
2020
ISSN:
0962-9351
Page Range / eLocation ID:
1 to 29
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; ; (, Nanomaterials)
    Inflammation, as induced by the presence of cytokines and chemokines, is an integral part of chlamydial infections. The anti-inflammatory cytokine, interleukin (IL)-10, has been reported to efficiently suppress the secretion of inflammatory cytokines triggered by Chlamydia in mouse macrophages. Though IL-10 is employed in clinical applications, its therapeutic usage is limited due to its short half-life. Here, we document the successful encapsulation of IL-10 within the biodegradable polymeric nanoparticles of PLA-PEG (Poly (lactic acid)-Poly (ethylene glycol), to prolong its half-life. Our results show the encapsulated-IL-10 size (~238 nm), zeta potential (−14.2 mV), polydispersity index (0.256), encapsulation efficiency (~77%), and a prolonged slow release pattern up to 60 days. Temperature stability of encapsulated-IL-10 was favorable, demonstrating a heat capacity of up to 89 °C as shown by differential scanning calorimetry analysis. Encapsulated-IL-10 modulated the release of IL-6 and IL-12p40 in stimulated macrophages in a time- and concentration-dependent fashion, and differentially induced SOCS1 and SOCS3 as induced by chlamydial stimulants in macrophages. Our finding offers the tremendous potential for encapsulated-IL-10 not only for chlamydial inflammatory diseases but also biomedical therapeutic applications. 
    more » « less
  2. ; ; ; ; (, Frontiers in Immunology)
    Resolution of inflammation is essential for normal tissue healing and regeneration, with macrophages playing a key role in regulating this process through phenotypic changes from a pro-inflammatory to an anti-inflammatory state. Pharmacological and mechanical (mechanotherapy) techniques can be employed to polarize macrophages toward an anti-inflammatory phenotype, thereby diminishing inflammation. One clinically relevant pharmacological approach is the inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4). This study investigates the effects of various mechanical loading amplitudes (0%, 3%, and 6%) and TRPV4 inhibition (10 µM RN-1734) on the phenotypic commitments of pro-inflammatory (M1) macrophages within three-dimensional (3D) collagen matrices. M1 macrophages exposed to 3% mechanical strain exhibited upregulated pro-inflammatory responses, including increased pro-inflammatory gene expression and enhanced proteolytic activity within the extracellular matrix. TRPV4 inhibition partially mitigated this inflammation. Notably, 6% mechanical strain combined with TRPV4 inhibition suppressed Mitogen-Activated Protein Kinase (MAPK) expression, leading to reduced pro-inflammatory gene expression and increased anti-inflammatory markers such as CD206. Gene expression analysis further demonstrated significant reductions in pro-inflammatory gene expression and a synergistic promotion of anti-inflammatory phenotypes under TRPV4 inhibition at 6% mechanical strain. Surface protein analysis via immunohistochemistry confirmed these phenotypic shifts, highlighting changes in the expression of CD80 (pro-inflammatory) and CD206 (anti-inflammatory) markers, alongside F-actin and nuclear staining. This research suggests that TRPV4 inhibition, combined with specific mechanical loading (6%), can drive macrophages toward an anti-inflammatory state, thereby may promote inflammation resolution and tissue repair. 
    more » « less
  3. ; ; ; (, Microorganisms)
    The type III secretion system (T3SS) effector EseN is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase activity. Previously we demonstrated that E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Comparative transcriptomic analysis showed a total of 753 significant differentially expressed genes in head-kidney-derived macrophages (HKDM) infected with an EseN mutant (∆EseN) compared to HKDM infected with wild-type (WT) strains. This data strongly indicates classical activation of macrophages (the M1 phenotype) in response to E. ictaluri infection and a significant role for EseN in the manipulation of this process. Our data also indicates that E. ictaluri EseN is involved in the modulation of pathways involved in the immune response to infection and expression of several transcription factors, including NF-κβ (c-rel and relB), creb3L4, socs6 and foxo3a. Regulation of transcription factors leads to regulation of proinflammatory interleukins (IL-8, IL-12a, IL-15, IL-6) and cyclooxygenase-2 (COX-2) expression. Inhibition of COX-2 mRNA by WT E. ictaluri leads to decreased production of prostaglandin E2 (PGE2), which is the product of COX-2 activity. Collectively, our results indicate that E. ictaluri EseN is an important player in the modulation of host immune responses to E.ictaluri infection. 
    more » « less
  4. ; ; ; ; ; ; ; ; ; ; et al (, APL Bioengineering)
    Cardiac adaptation to hypoxic injury is regulated by dynamic interactions between cardiomyocytes and macrophages, yet the impacts of immune phenotypes on cardiac structure and contractility remain poorly understood. To address this, we developed the immuno-heart on a chip, a novel in vitro platform to investigate cardiomyocyte–macrophage interactions under normoxic and hypoxic conditions. By integrating neonatal rat ventricular myocytes (NRVMs) and bone marrow-derived macrophages—polarized to pro-inflammatory (M1) or pro-healing (M2/M2*) phenotypes—we elucidated the dual protective and detrimental roles macrophages play in modulating cardiomyocyte cytoskeletal architecture and contractility. Pro-inflammatory stimulation reduced cardiomyocyte structural metrics (z-line length, fraction, and integrity) in normoxic co-cultures. Under hypoxia, M1-stimulated NRVM monocultures exhibited declines in cytoskeletal organization—quantified by actin and z-line orientational order parameters. Relative to monocultures, M1-stimulated co-cultures attenuated hypoxia-induced active stress declines but produced weaker normoxic stresses. In contrast, pro-healing stimulation improved normoxic z-line metrics and preserved post-hypoxia cytoskeletal organization but reduced normoxic contractility. Notably, M2-stimulated macrophages restored normoxic contractility and preserved post-hypoxia systolic stress, albeit with increased diastolic stress. RNAseq analysis of M2-stimulated co-cultures identified upregulated structural and immune pathways driving these hypoxia-induced changes. Cytokine profiles revealed stimulation-specific and density-dependent tumor necrosis factor-alpha and interleukin-10 secretion patterns. Together, these findings quantitatively link clinically relevant macrophage phenotypes and cytokines to distinct changes in cardiac structure and contractility, offering mechanistic insights into immune modulation of hypoxia-induced dysfunction. Moreover, the immuno-heart on a chip represents an innovative framework to guide the development of future therapies that integrate immune and cardiac targets to enhance patient outcomes. 
    more » « less
  5. ; ; ; ; ; ; ; (, Journal of Bacteriology)
    ABSTRACT Members of Chlamydia are obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other. Chlamydia spp. have five uncharacterized clp genes, clpX , clpC , two clpP paralogs, and clpB . In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactive clpP mutants in Chlamydia spp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detected in vitro . This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen. IMPORTANCE Chlamydia trachomatis is the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression in Chlamydia spp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email