Description: Regulatory T cells (Treg) are important in maintaining immune homeostasis and in regulating a variety of immune responses, making them attractive targets for modulating immune-related diseases. Success in using induction or transfer of Treg in mice to mediate transplant tolerance suggests Treg-based therapies as mechanisms of long-term drug-free transplant tolerance in human patients. Although more work is needed, critical analyses suggest that key factors in Treg induction, migration, and function are important areas to concentrate investigative efforts and therapeutic development. Elucidation of basic biology will aid in translating data gleaned from mice to humans so that Treg therapies become a reality for patients.

Description: Persistent activation of macrophages in lungs plays a critical role in the production of matrix metalloproteinases (MMPs) that contributes to the destruction of alveolar walls, a hallmark for pulmonary emphysema. Dysregulated TGF-β1 signaling has been an essential determinant in the elevation of MMPs during the development of emphysema. Nevertheless, the mechanism for this MMP-dependent pathogenesis has yet to be clearly investigated. Recently, we identified an important role for tyrosine phosphatase Src homology domain-containing protein tyrosine phosphatase 2 (Shp2) in regulating the activation of alveolar macrophages. Over a long-term observation period, mice with Shp2 deletion in macrophages ( LysMCre:Shp2 fl/fl ) develop spontaneous, progressive emphysema-like injury in the lungs, characterized by massive destruction of alveolar morphology, interstitial extracellular matrix degradation, and elevated levels of MMPs, particularly, significant increases of macrophage elastase (MMP12) in aged mice. Further analysis demonstrated that MMP12 suppression by TGF-β1 activation was apparently abrogated in LysMCre:Shp2 fl/fl mice, whereas the TGF-β1 concentration in the lungs was relatively the same. Mechanistically, we found that loss of Shp2 resulted in attenuated SMAD2/3 phosphorylation and nuclear translocation in response to TGF-β activation, thereby upregulating MMP12 expression in macrophages. Together, our findings define a novel physiological function of Shp2 in TGF-β1/MMP12-dependent emphysema, adding insights into potential etiologies for this chronic lung disorder.

Description: We have previously reported that a group of host cellular microRNAs (miRNAs; miR-34a-5p, miR-122-5p, miR-145-5p, miR-146a-5p, miR-210-3p) are released into the blood during sepsis, some of which are capable of inducing complement activation, cytokine production, and leukocyte migration. Extracellular vesicles (EVs) have been proposed as vehicles for extracellular miRNA-mediated intercellular communication. However, the biological function of plasma EVs and the associated miRNAs in sepsis are largely unknown. In this study, we tested the hypothesis that plasma EVs in sepsis are proinflammatory and EV-associated miRNAs are responsible for EV-induced cytokine production. Compared with those of sham mice, the plasma EVs from septic mice were slightly smaller (157 ± 2 versus 191 ± 6 nm, p 〈 0.0001), but more abundant [(1.6 ± 0.14) x 10 10 versus (0.93 ± 0.14) x 10 10 /ml plasma, p 〈 0.003]. miRNA array revealed that among 65 miRNAs, 8 miRNAs exhibited 〉1.5-fold increase in septic EVs compared with sham EVs, including miR-126-3p, miR-122-5p, miR-146a-5p, miR-145-5p, miR-26a-5p, miR-150-5p, miR-222-3p, and miR-181a-5p. Septic but not sham EVs were proinflammatory, promoting IL-6, TNF-α, IL-1β, and MIP-2 production. The effects of EVs were resistant to polymyxin B (an endotoxin inhibitor) but significantly inhibited by anti-miR inhibitors against miR-34a, miR-122, and miR-146a. Moreover, the septic EV-induced cytokine production was attenuated in TLR7 –/– or MyD88 –/– cells but remained the same in TLR3 –/– or Trif –/– cells. In vivo, mice i.p. injected with septic EVs had marked peritoneal neutrophil migration, which was significantly attenuated in MyD88 –/– mice. Taken together, these data demonstrate that plasma EVs of septic animals play an important role in inflammation, and EV-associated miRNAs likely mediate the cytokine production via TLR7-MyD88 signaling.

Description: Analysis of Ag-specific CD4 + T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. Although several methods exist for identifying Ag-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC class II multimers, all of these have significant technical constraints that limit their usefulness. Measurement of activation-induced expression of CD154 has been reported to detect live Ag-specific CD4 + T cells, but this approach remains underexplored and, to our knowledge, has not previously been applied in mycobacteria-infected animals. In this article, we show that CD154 expression identifies adoptively transferred or endogenous Ag-specific CD4 + T cells induced by Mycobacterium bovis bacillus Calmette-Guérin vaccination. We confirmed that Ag-specific cytokine production was positively correlated with CD154 expression by CD4 + T cells from bacillus Calmette-Guérin–vaccinated mice and show that high-quality microarrays can be performed from RNA isolated from CD154 + cells purified by cell sorting. Analysis of microarray data demonstrated that the transcriptome of CD4 + CD154 + cells was distinct from that of CD154 – cells and showed major enrichment of transcripts encoding multiple cytokines and pathways of cellular activation. One notable finding was the identification of a previously unrecognized subset of mycobacteria-specific CD4 + T cells that is characterized by the production of IL-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live Ag-specific CD4 + T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts.

Description: Chemotherapy-induced peripheral neuropathy (CIPN) is a painful and debilitating side effect of cancer chemotherapy with an unclear pathogenesis. Consequently, the available therapies for this neuropathic pain syndrome are inadequate, leading to a significantly reduced quality of life in many patients. Complement, a key component of the innate immune system, has been associated with neuroinflammation, a potentially important trigger of some types of neuropathic pain. However, the role of complement in CIPN remains unclear. To address this issue, we developed a C3 knockout (KO) rat model and induced CIPN in these KO rats and wild-type littermates via the i.p. administration of paclitaxel, a chemotherapeutic agent associated with CIPN. We then compared the severity of mechanical allodynia, complement activation, and intradermal nerve fiber loss between the groups. We found that 1) i.p. paclitaxel administration activated complement in wild-type rats, 2) paclitaxel-induced mechanical allodynia was significantly reduced in C3 KO rats, and 3) the paclitaxel-induced loss of intradermal nerve fibers was markedly attenuated in C3 KO rats. In in vitro studies, we found that paclitaxel-treated rat neuronal cells activated complement, leading to cellular injury. Our findings demonstrate a previously unknown but pivotal role of complement in CIPN and suggest that complement may be a new target for the development of novel therapeutics to manage this painful disease.

Description: Steroid receptor coactivator 3 (SRC-3) is a transcriptional coactivator that interacts with nuclear receptors and some other transcription factors to enhance their effects on target gene transcription. We reported previously that SRC-3–deficient (SRC-3 –/– ) mice are extremely susceptible to Escherichia coli– induced septic peritonitis as a result of uncontrolled inflammation and a defect in bacterial clearance. In this study, we observed significant upregulation of SRC-3 in colonic epithelial cells in response to Citrobacter rodentium infection. Based on these findings, we hypothesized that SRC-3 is involved in host defense against attaching and effacing bacterial infection. We compared the responses of SRC-3 –/– and wild-type mice to intestinal C. rodentium infection. We found that SRC-3 –/– mice exhibited delayed clearance of C. rodentium and more severe tissue pathology after oral infection with C. rodentium compared with wild-type mice. SRC-3 –/– mice expressed normal antimicrobial peptides in the colons but exhibited delayed recruitment of neutrophils into the colonic mucosa. Accordingly, SRC-3 –/– mice showed a delayed induction of CXCL2 and CXCL5 in colonic epithelial cells, which are responsible for neutrophil recruitment. At the molecular level, we found that SRC-3 can activate the NF-B signaling pathway to promote CXCL2 expression at the transcriptional level. Collectively, we show that SRC-3 contributes to host defense against enteric bacteria, at least in part via upregulating CXCL2 expression to recruit neutrophils.

Description: The recognition of pathogen-associated molecular patterns is accomplished by the recognition modules of pattern recognition receptors (PRRs). Leucine-rich repeats (LRRs) and C-type lectin-like domain (CTLD) represent the two most universal categories of recognition modules. In the current study, we identified a novel soluble and bacteria-inducible PRR comprising LRRs and a CTLD from the hepatopancreas of kuruma shrimp Marsupenaeus japonicus and named it Leulectin. The module arrangement of Leulectin is unique among all organisms. Both modules, together with the whole molecule, protected shrimp against Vibrio infection. By screening the pathogen-associated molecular patterns that shrimp might encounter, Leulectin was found to sense Vibrio flagellin through the LRRs and to recognize LPS through CTLD. The LRR–flagellin interaction was confirmed by pull-down and far-Western assays and was found to rely on the fourth LRR of Leulectin and the N terminus of flagellin. The recognition of LPS was determined by the long loop region of CTLD in a calcium-independent manner. By sensing the flagellin, LRRs could prevent its attachment to shrimp cells, thereby inhibiting Vibrio colonization. With the ability to recognize LPS, CTLD could agglutinate the bacteria and promote hemocytic phagocytosis. Our study clearly showed the division of labor and the synergy between different recognition modules and provided new insights into the concept of pattern recognition and the function of soluble PRRs in the antibacterial response.

Description: The aim of this study was to elucidate the expression and functions of IL-24 in C57BL/6 mouse corneas in response to Pseudomonas aeruginosa infection. Among IL-20R cytokines, only IL-24 was induced at both mRNA and protein levels by infection at early time points. The upregulation of IL-24 was dampened by flagellin pretreatment, which protects the corneas from microbial infection. Time course studies revealed bimodal early and later peaks of IL-24 expression, a pattern shared with suppressor of cytokine signaling (SOCS)3 but not IL-1β or IL-6. Silencing of IL-24 enhanced S100A8/A9 expression and suppressed SOCS3, IL-1β, IL-1RN, and matrix metalloproteinase 13 expression at 6 h postinfection. Downregulation of the IL-24 signaling pathway significantly reduced the severity of keratitis, whereas rIL-24 exacerbated P. aeruginosa –mediated tissue destruction. In vitro, rIL-1β induced the expression of SOCS3, IL-24, IL-1β, and IL-6 in primary cultured human corneal epithelial cells. rIL-24, alternatively, stimulated the expression of SOCS3, but not the others. In conclusion, IL-24 promotes P. aeruginosa keratitis through the suppression of early protective mucosal immunity, culminating in increased severity of P. aeruginosa keratitis.

Description: The scavenger receptor macrophage receptor with collagenous structure (MARCO) promotes protective innate immunity against bacterial and parasitic infections; however, its role in host immunity against fungal pathogens, including the major human opportunistic fungal pathogen Cryptococcus neoformans , remains unknown. Using a mouse model of C. neoformans infection, we demonstrated that MARCO deficiency leads to impaired fungal control during the afferent phase of cryptococcal infection. Diminished fungal containment in MARCO –/– mice was accompanied by impaired recruitment of Ly6C high monocytes and monocyte-derived dendritic cells (moDC) and lower moDC costimulatory maturation. The reduced recruitment and activation of mononuclear phagocytes in MARCO –/– mice was linked to diminished early expression of IFN- along with profound suppression of CCL2 and CCL7 chemokines, providing evidence for roles of MARCO in activation of the CCR2 axis during C. neoformans infection. Lastly, we found that MARCO was involved in C. neoformans phagocytosis by resident pulmonary macrophages and DC. We conclude that MARCO facilitates early interactions between C. neoformans and lung-resident cells and promotes the production of CCR2 ligands. In turn, this contributes to a more robust recruitment and activation of moDC that opposes rapid fungal expansion during the afferent phase of cryptococcal infection.

Description: IL-35 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p35 subunit and an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3). IL-35 functions through IL-35R and has a potent immune-suppressive activity. Although IL-35 was demonstrated to be produced by regulatory T cells, gene-expression analysis revealed that it is likely to have a wider distribution, including expression in cancer cells. In this study, we demonstrated that IL-35 is produced in human cancer tissues, such as large B cell lymphoma, nasopharyngeal carcinoma, and melanoma. To determine the roles of tumor-derived IL-35 in tumorigenesis and tumor immunity, we generated IL-35–producing plasmacytoma J558 and B16 melanoma cells and observed that the expression of IL-35 in cancer cells does not affect their growth and survival in vitro, but it stimulates tumorigenesis in both immune-competent and Rag1/2-deficient mice. Tumor-derived IL-35 increases CD11b + Gr1 + myeloid cell accumulation in the tumor microenvironment and, thereby, promotes tumor angiogenesis. In immune-competent mice, spontaneous CTL responses to tumors are diminished. IL-35 does not directly inhibit tumor Ag–specific CD8 + T cell activation, differentiation, and effector functions. However, IL-35–treated cancer cells had increased expression of gp130 and reduced sensitivity to CTL destruction. Thus, our study indicates novel functions for IL-35 in promoting tumor growth via the enhancement of myeloid cell accumulation, tumor angiogenesis, and suppression of tumor immunity.