Yongliang Zhang

STAT5 programs a distinct subset of GM-CSF-producing T helper cells that is essential for autoimmune neuroinflammation

T helper (TH)-cell subsets, such as TH1 and TH17, mediate inflammation in both peripheral tissues and central nervous system. Here we show that STAT5 is required for T helper-cell pathogenicity in autoimmune neuroinflammation but not in experimental colitis. Although STAT5 promotes regulatory T cell generation and immune suppression, loss of STAT5 in CD4+ T cells resulted in diminished development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Our results showed that loss of encephalitogenic activity of STAT5-deficient autoreactive CD4+ T cells was independent of IFN-γ or interleukin 17 (IL-17) production, but was due to the impaired expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), a crucial mediator of T-cell pathogenicity. We further showed that IL-7-activated STAT5 promotes the generation of GM-CSF-producing CD4+ T cells, which were preferentially able to induce more severe EAE than TH17 or TH1 cells. Consistent with GM-CSF-producing cells being a distinct subset of TH cells, the differentiation program of these cells was distinct from that of TH17 or TH1 cells. We further found that IL-3 was secreted in a similar pattern as GM-CSF in this subset of TH cells. In conclusion, the IL-7-STAT5 axis promotes the generation of GM-CSF/IL-3-producing TH cells. These cells display a distinct transcriptional profile and may represent a novel subset of T helper cells which we designate as TH-GM.

The fate and lifespan of human monocyte subsets in steady state and systemic inflammation

In humans, the monocyte pool comprises three subsets (classical, intermediate, and nonclassical) that circulate in dynamic equilibrium. The kinetics underlying their generation, differentiation, and disappearance are critical to understanding both steady-state homeostasis and inflammatory responses. Here, using human in vivo deuterium labeling, we demonstrate that classical monocytes emerge first from marrow, after a postmitotic interval of 1.6 d, and circulate for a day. Subsequent labeling of intermediate and nonclassical monocytes is consistent with a model of sequential transition. Intermediate and nonclassical monocytes have longer circulating lifespans (∼4 and ∼7 d, respectively). In a human experimental endotoxemia model, a transient but profound monocytopenia was observed; restoration of circulating monocytes was achieved by the early release of classical monocytes from bone marrow. The sequence of repopulation recapitulated the order of maturation in healthy homeostasis. This developmental relationship between monocyte subsets was verified by fate mapping grafted human classical monocytes into humanized mice, which were able to differentiate sequentially into intermediate and nonclassical cells.

MAPK Phosphatase 5 Expression Induced by Influenza and Other RNA Virus Infection Negatively Regulates IRF3 Activation and Type I Interferon Response

The type I interferon system is essential for antiviral immune response and is a primary target of viral immune evasion strategies. Here, we show that virus infection induces the expression of MAPK phosphatase 5 (MKP5), a dual-specificity phosphatase (DUSP), in host cells. Mice deficient in MKP5 were resistant to H1N1 influenza infection, which is associated with increased IRF3 activation and type I interferon expression in comparison with WT mice. Increased type I interferon responses were also observed in MKP5-deficient cells and animals upon other RNA virus infection, including vesicular stomatitis virus and sendai virus. These observations were attributed to the ability of MKP5 to interact with and dephosphorylate IRF3. Our study reveals a critical function of a DUSP in negative regulation of IRF3 activity and demonstrates a mechanism by which influenza and other RNA viruses inhibit type I interferon response in the host through MKP5.

Differential Regulation of Proinflammatory Cytokine Expression by Mitogen-Activated Protein Kinases in Macrophages in Response to Intestinal Parasite Infection

ABSTRACT Blastocystis is a common enteric protistan parasite that can cause acute, as well as chronic, infection and is associated with irritable bowel syndrome (IBS). However, the pathogenic status of Blastocystis infection remains unclear. In this study, we found that Blastocystis antigens induced abundant expression of proinflammatory cytokines, including interleukin 1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), in mouse intestinal explants, in mouse colitis colon, and in macrophages. Further investigation utilizing RAW264.7 murine macrophages showed that Blastocystis treatment in RAW264.7 macrophages induced the activation of ERK, JNK, and p38, the three major groups of mammalian mitogen-activated protein (MAP) kinases that play essential roles in the expression of proinflammatory cytokines. ERK inhibition in macrophages significantly suppressed both mRNA and protein expression of IL-6 and TNF-α and mRNA expression of IL-1β. On the other hand, JNK inhibition resulted in reductions in both c-Jun and ERK activation and significant suppression of all three proinflammatory cytokines at both the mRNA and protein levels. Inhibition of p38 suppressed only IL-6 protein expression with no effect on the expression of IL-1β and TNF-α. Furthermore, we found that serine proteases produced by Blastocystis play an important role in the induction of ERK activation and proinflammatory cytokine expression by macrophages. Our study thus demonstrated for the first time that Blastocystis could induce the expression of various proinflammatory cytokines via the activation of MAP kinases and that infection with Blastocystis may contribute to the pathogenesis of inflammatory intestinal diseases through the activation of inflammatory pathways in host immune cells, such as macrophages.

Cd47-Sirpα interaction and IL-10 constrain inflammation-induced macrophage phagocytosis of healthy self-cells

Significance The present study reveals that macrophage phagocytosis toward healthy self-cells is controlled by a two-tier mechanism: a forefront activation mechanism requiring the inflammatory cytokine-stimulated protein kinase C (PKC)-spleen tyrosine kinase (Syk) pathway, to which IL-10 conversely regulates, and the subsequent self-target discrimination mechanism controlled by the CD47-signal regulatory protein α (SIRPα)–mediated inhibition. The findings significantly expand our understanding of macrophage phagocytic plasticity and behavior under different conditions and also provide insights into strategies for enhancing transplantation tolerance and macrophage-based cancer eradication, especially for cancers toward which therapeutic antibodies are yet unavailable. Rapid clearance of adoptively transferred Cd47-null (Cd47−/−) cells in congeneic WT mice suggests a critical self-recognition mechanism, in which CD47 is the ubiquitous marker of self, and its interaction with macrophage signal regulatory protein α (SIRPα) triggers inhibitory signaling through SIRPα cytoplasmic immunoreceptor tyrosine-based inhibition motifs and tyrosine phosphatase SHP-1/2. However, instead of displaying self-destruction phenotypes, Cd47−/− mice manifest no, or only mild, macrophage phagocytosis toward self-cells except under the nonobese diabetic background. Studying our recently established Sirpα-KO (Sirpα−/−) mice, as well as Cd47−/− mice, we reveal additional activation and inhibitory mechanisms besides the CD47-SIRPα axis dominantly controlling macrophage behavior. Sirpα−/− mice and Cd47−/− mice, although being normally healthy, develop severe anemia and splenomegaly under chronic colitis, peritonitis, cytokine treatments, and CFA-/LPS-induced inflammation, owing to splenic macrophages phagocytizing self-red blood cells. Ex vivo phagocytosis assays confirmed general inactivity of macrophages from Sirpα−/− or Cd47−/− mice toward healthy self-cells, whereas they aggressively attack toward bacteria, zymosan, apoptotic, and immune complex-bound cells; however, treating these macrophages with IL-17, LPS, IL-6, IL-1β, and TNFα, but not IFNγ, dramatically initiates potent phagocytosis toward self-cells, for which only the Cd47-Sirpα interaction restrains. Even for macrophages from WT mice, phagocytosis toward Cd47−/− cells does not occur without phagocytic activation. Mechanistic studies suggest a PKC-Syk–mediated signaling pathway, to which IL-10 conversely inhibits, is required for activating macrophage self-targeting, followed by phagocytosis independent of calreticulin. Moreover, we identified spleen red pulp to be one specific tissue that provides stimuli constantly activating macrophage phagocytosis albeit lacking in Cd47−/− or Sirpα−/− mice.

Casein Kinase II Controls TBK1/IRF3 Activation in IFN Response against Viral Infection

By sensing viral nucleic acids, host innate receptors elicit signaling pathways converging on TBK1-IFN regulatory factor (IRF)3 axis in mediating IFN-αβ induction and defense mechanisms. In contrast, viruses have evolved with diverse immune evasion/interference mechanisms to undermine innate receptor signaling and IFN response. In this regard, approaches enabling host to overcome such immune evasion/interference mechanisms are urgently needed to combat infections by epidemic/pandemic viruses. In this study, we report that protein kinase CK2 serves as a key component controlling TBK1 and IRF3 activation in IFN-inducing TLR, RIG-I–like receptors, and cGAS/STING signaling pathways. Accordingly, knocking down of CK2 expression or genetic ablation of its kinase activity resulted in elevated IFN-αβ response in response to infection by DNA and RNA viruses. Moreover, PP2A was identified as one of the intermediate phosphatases responsible for CK2-regulated IFN response, suggesting that CK2 may regulate TBK1 and IRF3 activation indirectly. Importantly, blockade of CK2 activity by small molecule inhibitor was able to activate TBK1, whereby eliciting effective host defense mechanisms against hepatitis C virus infection. Taken together, our results identify CK2 as a novel regulator of TBK1 and IRF3 and suggest that targeting CK2 by small molecular inhibitor may be a viable approach to prevent and treat viral infections.

DUSP10 regulates intestinal epithelial cell growth and colorectal tumorigenesis

Dual specificity phosphatase 10 (DUSP10), also known as MAP kinase phosphatase 5 (MKP5), negatively regulates the activation of MAP kinases. Genetic polymorphisms and aberrant expression of this gene are associated with colorectal cancer (CRC) in humans. However, the role of DUSP10 in intestinal epithelial tumorigenesis is not clear. Here, we showed that DUSP10 knockout (KO) mice had increased intestinal epithelial cell (IEC) proliferation and migration and developed less severe colitis than wild-type (WT) mice in response to dextran sodium sulphate (DSS) treatment, which is associated with increased ERK1/2 activation and Krüppel-like factor 5 (KLF5) expression in IEC. In line with increased IEC proliferation, DUSP10 KO mice developed more colon tumours with increased severity compared with WT mice in response to administration of DSS and azoxymethane (AOM). Furthermore, survival analysis of CRC patients demonstrated that high DUSP10 expression in tumours was associated with significant improvement in survival probability. Overexpression of DUSP10 in Caco-2 and RCM-1 cells inhibited cell proliferation. Our study showed that DUSP10 negatively regulates IEC growth and acts as a suppressor for CRC. Therefore, it could be targeted for the development of therapies for colitis and CRC.

Capsules of virulent pneumococcal serotypes enhance formation of neutrophil extracellular traps during in vivo pathogenesis of pneumonia

Neutrophil extracellular traps (NETs) are released by activated neutrophils to ensnare and kill microorganisms. NETs have been implicated in tissue injury since they carry cytotoxic components of the activated neutrophils. We have previously demonstrated the generation of NETs in infected murine lungs during both primary pneumococcal pneumonia and secondary pneumococcal pneumonia after primary influenza. In this study, we assessed the correlation of pneumococcal capsule size with pulmonary NETs formation and disease severity. We compared NETs formation in the lungs of mice infected with three pneumococcal strains of varying virulence namely serotypes 3, 4 and 19F, as well as a capsule-deficient mutant of serotype 4. In primary pneumonia, NETs generation was strongly associated with the pneumococcal capsule thickness, and was proportional to the disease severity. Interestingly, during secondary pneumonia after primary influenza infection, intense pulmonary NETs generation together with elevated myeloperoxidase activity and cytokine dysregulation determined the disease severity. These findings highlight the crucial role played by the size of pneumococcal capsule in determining the extent of innate immune responses such as NETs formation that may contribute to the severity of pneumonia.

Beneficial innate signaling interference for antibacterial responses by a Toll-like receptor-mediated enhancement of the MKP-IRF3 axis.

Significance Infection by a given pathogen results in stimulation of multiple classes of innate receptors in a cell, leading to activation of distinct signaling pathways. However, these pathways are not always beneficial to immune response against the pathogen. This study shows that, on infection by pathogenic bacterium Listeria monocytogenes, Toll-like receptor (TLR) pathways suppress type I IFN gene induction that is mediated by stimulator of IFN genes. Type I IFNs are critical for antiviral immunity but detrimental to macrophage bactericidal activity. The TLR pathways selectively suppress IFN regulatory factor 3, an essential transcription factor for type I IFN gene induction, through induction/activation of mitogen-activated protein kinase phosphatases, revealing a unique mechanism of beneficial innate signaling interference against bacterial infection. A major function of innate immune receptors is to recognize pathogen-associated molecular patterns and then evoke immune responses appropriate to the nature of the invading pathogen(s). Because innate immune cells express various types of these receptors, distinct combinations of signaling pathways are activated in response to a given pathogen. Although the conventional wisdom is that these signaling pathways cooperate with one another to ensure an effective host response, a more nuanced view recognizes antagonism between the individual pathways, where the attenuation of a signaling pathway(s) by others may shape the immune response. In this study, we show that, on Listeria monocytogenes infection, Toll-like receptor-triggered MyD88 signaling pathways suppress type I IFN gene induction, which is detrimental to macrophage bactericidal activity. These pathways target and suppress the IFN regulatory factor 3 (IRF3) transcription factor that is activated by the stimulator of IFN genes–TANK-binding kinase-1 kinase pathway. We also provide evidence for the involvement of the MAPK phosphatase family members, which renders IRF3 hypophosphorylated on Toll-like receptor signaling by enhancing the formation of an MAPK phosphatase–IRF3–TANK-binding kinase-1 ternary complex. This study, therefore, reveals a hitherto unrecognized and important contribution of a beneficial innate signaling interference against bacterial infections.

Ex Vivo and In Vivo Mice Models to Study Blastocystis spp. Adhesion, Colonization and Pathology: Closer to Proving Koch's Postulates.

Blastocystis spp. are widely prevalent extra cellular, non-motile anerobic protists that inhabit the gastrointestinal tract. Although Blastocystis spp. have been associated with gastrointestinal symptoms, irritable bowel syndrome and urticaria, their clinical significance has remained controversial. We established an ex vivo mouse explant model to characterize adhesion in the context of tissue architecture and presence of the mucin layer. Using confocal microscopy with tissue whole mounts and two axenic isolates of Blastocystis spp., subtype 7 with notable differences in adhesion to intestinal epithelial cells (IEC), isolate B (ST7-B) and isolate H (more adhesive, ST7-H), we showed that adhesion is both isolate dependent and tissue trophic. The more adhesive isolate, ST7-H was found to bind preferentially to the colon tissue than caecum and terminal ileum. Both isolates were also found to have mucinolytic effects. We then adapted a DSS colitis mouse model as a susceptible model to study colonization and acute infection by intra-caecal inoculation of trophic Blastocystis spp.cells. We found that the more adhesive isolate ST7-H was also a better colonizer with more mice shedding parasites and for a longer duration than ST7-B. Adhesion and colonization was also associated with increased virulence as ST7-H infected mice showed greater tissue damage than ST7-B. Both the ex vivo and in vivo models used in this study showed that Blastocystis spp. remain luminal and predominantly associated with mucin. This was further confirmed using colonic loop experiments. We were also successfully able to re-infect a second batch of mice with ST7-H isolates obtained from fecal cultures and demonstrated similar histopathological findings and tissue damage thereby coming closer to proving Koch’s postulates for this parasite.