Irina N. Shalova

Characterization of the nature of granulocytic myeloid-derived suppressor cells in tumor-bearing mice.

MDSCs are a group of cells with potent immune‐suppressive activity. These cells accumulate in many pathologic conditions and play a major role in the regulation of immune responses. The nature of MDSC remains highly debatable. In cancer, most MDSCs are represented by cells with granulocytic phenotype and morphology, G‐MDSC. The relationship between G‐MDSCs and Neu remains unclear. In this study, we have found that G‐MDSCs, from tumor‐bearing, and Neu, from tumor‐free, mice share a common morphology and phenotype. However, in contrast to Neu, a substantial proportion of G‐MDSCs expressed M‐CSFR and a CD244 molecule. Neu had significantly higher phagocytic activity, expression of lysosomal proteins, and TNF‐α than corresponding G‐MDSCs, which had significantly higher activity of arginase, MPO, and ROS. In contrast to G‐MDSC, neither rested nor mobilized Neu suppressed T cells. G‐MDSC survived 2 days in culture in the presence of GM‐CSF and within 24 h, became phenotypic and functionally similar to Neu. Tumor‐associated G‐MDSC shared most characteristics of splenic G‐MDSC, rather then Neu. These data suggest that in cancer, despite morphological and phenotypic similarities, G‐MDSCs are functionally distinct from Neu and are comprised of pathologically activated precursors of Neu.

Macrophage polarization and plasticity in health and disease.

The role of myelomonocytic cells like monocytes and macrophages as first line of host defense is well established. Recent understanding of these cells using systems biology, transgenesis and in disease models has brought them to a center stage in orchestrating crucial functions during homeostasis and pathogenesis. Thus, understanding the functional diversity of these cells in health and disease as well as the mechanisms that control these events would be crucial for designing strategies for regulating disease and reinstate homeostasis.

Macrophage polarization to a unique phenotype driven by B cells

Regulation of adaptive immunity by innate immune cells is widely accepted. Conversely, adaptive immune cells can also regulate cells of the innate immune system. Here, we report for the first time the essential role of B cells in regulating macrophage (Mϕ) phenotype. In vitro B cell/Mϕ co‐culture experiments together with experiments in transgenic mice models for B‐cell deficiency or overexpression showed B1 cells to polarize Mϕ to a distinct phenotype. This was characterized by downregulated TNF‐α, IL‐1β and CCL3, but upregulated IL‐10 upon LPS stimulation; constitutive expression of M2 Mϕ markers (e.g. Ym1, Fizz1) and overexpression of TRIF‐dependent cytokines (IFN‐β, CCL5). Mechanistically, this phenotype was linked to a defective NF‐κB activation, but a functional TRIF/STAT1 pathway. B1‐cell‐derived IL‐10 was found to be instrumental in the polarization of these Mϕ. Finally, in vivo relevance of B1‐cell‐induced Mϕ polarization was confirmed using the B16 melanoma tumor model where adoptive transfer of B1 cells induced an M2 polarization of tumor‐associated Mϕ. Collectively, our results define a new mechanism of Mϕ polarization wherein B1 cells play a key role in driving Mϕ to a unique, but M2‐biased phenotype. Future studies along these lines may lead to targeting of B1 cells to regulate Mϕ response in inflammation and cancer.

Human Monocytes Undergo Functional Re-programming during Sepsis Mediated by Hypoxia-Inducible Factor-1α

Sepsis is characterized by a dysregulated inflammatory response to infection. Despite studies in mice, the cellular and molecular basis of human sepsis remains unclear and effective therapies are lacking. Blood monocytes serve as the first line of host defense and are equipped to recognize and respond to infection by triggering an immune-inflammatory response. However, the response of these cells in human sepsis and their contribution to sepsis pathogenesis is poorly understood. To investigate this, we performed a transcriptomic, functional, and mechanistic analysis of blood monocytes from patients during sepsis and after recovery. Our results revealed the functional plasticity of monocytes during human sepsis, wherein they transited from a pro-inflammatory to an immunosuppressive phenotype, while enhancing protective functions like phagocytosis, anti-microbial activity, and tissue remodeling. Mechanistically, hypoxia inducible factor-1α (HIF1α) mediated this functional re-programming of monocytes, revealing a potential mechanism for their therapeutic targeting to regulate human sepsis.

CD16 Regulates TRIF-Dependent TLR4 Response in Human Monocytes and Their Subsets

Blood monocytes recognize Gram-negative bacteria through the TLR4, which signal via MyD88- and TRIF-dependent pathway to trigger an immune-inflammatory response. However, a dysregulated inflammatory response by these cells often leads to severe pathologies such as sepsis. We investigated the role of CD16 in the regulation of human monocyte response to Gram-negative endotoxin and sepsis. Blood monocytes from sepsis patients demonstrated an upregulation of several TRIF-dependent genes as well as a selective expansion of CD16-expressing (CD16+) monocytes. Gene expression and biochemical studies revealed CD16 to regulate the TRIF-dependent TLR4 pathway in monocytes by activating Syk, IFN regulatory factor 3, and STAT1, which resulted in enhanced expression of IFNB, CCL5, and CXCL10. CD16 also upregulated the expression of IL-1R–associated kinase M and IL-1 receptor antagonist, which are negative regulators of the MyD88-dependent pathway. CD16 overexpression or small interfering RNA knockdown in monocytes confirmed the above findings. Interestingly, these results were mirrored in the CD16+ monocyte subset isolated from sepsis patients, providing an in vivo confirmation to our findings. Collectively, the results from the current study demonstrate CD16 as a key regulator of the TRIF-dependent TLR4 pathway in human monocytes and their CD16-expressing subset, with implications in sepsis.

Interferon-γ and Granulocyte/Monocyte Colony-stimulating Factor Production by Natural Killer Cells Involves Different Signaling Pathways and the Adaptor Stimulator of Interferon Genes (STING)

Background: NK produce IFN-γ and GM-CSF in response to CpG-ODN in the presence of accessory cytokines. Results: This production involves NF-κB, STAT3, UNC93b1 and IL-12. IFN-γ is MyD88- and TLR9-dependent, whereas GM-CSF depends on the adaptor STING. Conclusion: NK present an alternative mechanism of sensing of CpG-ODN. Significance: These results open new horizons in the understanding of NK cells activation by microbial DNA. Natural killer (NK) cells are important for innate immunity in particular through the production of IFN-γ and GM-CSF. Both cytokines are important in restoration of immune function of tolerized leukocytes under inflammatory events. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors, rarely seeking their protein expression. We previously showed that murine spleen NK cells express TLR9 intracellularly and respond to CpG oligodeoxynucleotide (CpG-ODN) by producing IFN-γ and GM-CSF. However, to get such production the presence of accessory cytokines (such as IL-15 and IL-18) was required, whereas CpG-ODN or accessory cytokines alone did not induce IFN-γ or GM-CSF. We show here that TLR9 overlaps with the Golgi apparatus in NK cells. Furthermore, CpG-ODN stimulation in the presence of accessory cytokines induces the phosphorylation of c-Jun, STAT3, and IκBα. IFN-γ and GM-CSF production requires NF-κB and STAT3 activation as well as Erk-dependent mechanisms for IFN-γ and p38 signaling for GM-CSF. Using knock-out-mice, we show that UNC93b1 and IL-12 (produced by NK cells themselves) are also necessary for IFN-γ and GM-CSF production. IFN-γ production was found to be MyD88- and TLR9-dependent, whereas GM-CSF was TLR9-independent but dependent on STING (stimulator of interferon genes), a cytosolic adaptor recently described for DNA sensing. Our study thereby allows us to gain insight into the mechanisms of synergy between accessory cytokines and CpG-ODN in NK cells. It also identifies a new and alternative signaling pathway for CpG-ODN in murine NK cells.

Metabolic regulation of macrophage phenotype and function

Studies in the last 20 years have given us a remarkable insight into the functional and phenotypic diversity of macrophages which reflects their integral role in host defence, homeostasis and pathogenesis. Mouse genetics, transcriptomic and epigenetic studies have provided an ontogenic and molecular perspective to the phenotypic diversity of these cells. Recently, metabolic studies have revealed the crucial role of metabolism and metabolites in shaping the phenotype and function of macrophages. Evidence pertaining to this aspect will be reviewed here.

Endotoxin Tolerance as a Key Mechanism for Immunosuppression

Inflammation is a complex pathophysiological phenomenon orchestrated by immune cells in response to infection and/or tissue damage (Nathan, 2002; Foster & Medzhitov, 2009). It serves protective mechanism against pathological insults and aims to re-instate homeostasis. Monocytes/macrophages are the first line of immune cells to detect and response to ‘‘danger signals’’ in an organism (e.g. pathogens, tissue damage). The detection of pathogens and/or endotoxins by these cells is mediated through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) which triggers a robust and inflammatory reaction (Figure 1). However, uncontrolled inflammation can lead to extensive tissue damage and manifestation of pathological states like sepsis, autoimmune diseases, metabolic diseases and cancer (Foster & Medzhitov, 2009). Thus, the innate immune cells ‘adapt’ themselves in the later phase of inflammation to tune down this response and promote resolution of inflammation leading to healing and tissue repair (Figure 1). Organisms as well as their immune cells have developed mechanisms to protect themselves from excessive inflammation in response to endotoxins. Endotoxin tolerance (ET) is such an adaptation wherein organisms or their innate immune cells (like monocytes/macrophages) show diminished response to endotoxins as a result of prior exposure to low doses of endotoxins (Foster & Medzhitov, 2009; Biswas et al., 2007; Dobrovolskaia & Vogel, 2002; Fan & Cook, 2004; Cavaillon & Adib-Conquy, 2006). In other words, the organism or their immune cells have developed a ’’tolerance” to endotoxin. Clinically, this phenomenon can be observed in monocytes/macrophages in patients with sepsis, trauma, surgery or pancreatitis (Cavaillon et al., 2003; Monneret et al., 2008). In most of these cases ET contributes to immunosuppression, while in sepsis it has been linked to mortality as well (Figure 1) (Monneret et al., 2008). These and other facts have suggested ET as a key mechanism for immunosuppression associated with diverse pathological conditions. In this chapter, we will review the in vitro and in vivo evidences for ET, as well as an insight into the cellular and molecular basis of this phenomenon. In addition, the pathophysiological implications of ET will be also discussed.