A systematic quantitative analysis of temporal changes in host and viral proteins throughout the course of a productive infection could provide dynamic insights into virus-host interaction. We developed a proteomic technique called "quantitative temporal viromics" (QTV), which employs multiplexed tandem-mass-tag-based mass spectrometry. Human cytomegalovirus (HCMV) is not only an important pathogen but a paradigm of viral immune evasion. QTV detailed how HCMV orchestrates the expression of >8,000 cellular proteins, including 1,200 cell-surface proteins to manipulate signaling pathways and counterintrinsic, innate, and adaptive immune defenses. QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets. Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined. QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model.

The successful resolution of inflammation is dependent upon the coordinated transition from the initial recruitment of neutrophils to a more sustained population of mononuclear cells. IL-6, which signals via the common receptor subunit gp130, represents a crucial checkpoint regulator of neutrophil trafficking during the inflammatory response by orchestrating chemokine production and leukocyte apoptosis. However, the relative contribution of specific IL-6-dependent signaling pathways to these processes remains unresolved. To define the receptor-mediated signaling events responsible for IL-6-driven neutrophil trafficking, we used a series of gp130 knockin mutant mice displaying altered IL-6-signaling capacities in an experimental model of acute peritoneal inflammation. Hyperactivation of STAT1 and STAT3 in gp130^Y757F/Y757F mice led to a more rapid clearance of neutrophils, and this coincided with a pronounced down-modulation in production of the neutrophil-attracting chemokine CXCL1/KC. By contrast, the proportion of apoptotic neutrophils in the inflammatory infiltrate remained unaffected. In gp130^Y757F/Y757F mice lacking IL-6, neutrophil trafficking and CXCL1/KC levels were normal, and this corresponded with a reduction in the level of STAT1/3 activity. Furthermore, monoallelic ablation of Stat3 in gp130^Y757F/Y757F mice specifically reduced STAT3 activity and corrected both the rapid clearance of neutrophils and impaired CXCL1/KC production. Conversely, genetic deletion of Stat1 in gp130^Y757F/Y757F mice failed to rescue the altered responses observed in gp130^Y757F/Y757F mice. Collectively, these data genetically define that IL-6-driven signaling via STAT3, but not STAT1, limits the inflammatory recruitment of neutrophils, and therefore represents a critical event for the termination of the innate immune response.

Fibrosis in response to tissue damage or persistent inflammation is a pathological hallmark of many chronic degenerative diseases. By using a model of acute peritoneal inflammation, we have examined how repeated inflammatory activation promotes fibrotic tissue injury. In this context, fibrosis was strictly dependent on interleukin-6 (IL-6). Repeat inflammation induced IL-6-mediated T helper 1 (Th1) cell effector commitment and the emergence of STAT1 (signal transducer and activator of transcription-1) activity within the peritoneal membrane. Fibrosis was not observed in mice lacking interferon-γ (IFN-γ), STAT1, or RAG-1. Here, IFN-γ and STAT1 signaling disrupted the turnover of extracellular matrix by metalloproteases. Whereas IL-6-deficient mice resisted fibrosis, transfer of polarized Th1 cells or inhibition of MMP activity reversed this outcome. Thus, IL-6 causes compromised tissue repair by shifting acute inflammation into a more chronic profibrotic state through induction of Th1 cell responses as a consequence of recurrent inflammation.

Interleukin (IL)-6 signaling through its soluble receptor (IL-6 transsignaling) directs transition between innate and acquired immune responses by orchestrating the chemokine-directed attraction and apoptotic clearance of leukocytes. Through analysis of mononuclear cell infiltration in WT and IL-6-deficient mice during peritoneal inflammation, we now report that IL-6 selectively governs T cell infiltration by regulating chemokine secretion (CXCL10, CCL4, CCL5, CCL11, and CCL17) and chemokine receptor (CCR3, CCR4, CCR5, and CXCR3) expression on the CD3⁺infiltrate. Although blockade of IL-6 trans-signaling prevented chemokine release, chemokine receptor expression remained unaltered suggesting that this response is regulated by IL-6 itself. To dissect the signaling events promoting T cell migration, inflammation was established in knock-in mice expressing mutated forms of the universal signal-transducing element for IL-6-related cytokines gp130. In mice (gp130^Y757F/Y757F) deficient in SHP2 and SOCS3 binding, but presenting hyperactivation of STAT1/3, T cell recruitment and CCL5 expression was enhanced. Conversely, both of these parameters were suppressed in mice with ablated gp130-mediated STAT1/3 activation (gp130^{ΔSTAT/ΔSTAT}). T cell migration was related to STAT3 activity, because monoallelic deletion ofStat3ingp130^Y757F/Y757Fmice (gp130^Y757F/Y757F:Stat3^+/-) corrected the exaggerated responses observed ingp130^Y757F/Y757Fmice. Consequently, STAT3 plays a defining role in IL-6-mediated T cell migration.

Interleukin 6 (IL6) trans-signaling has emerged as a prominent regulator of immune responses during both innate and acquired immunity. Regulation of IL6 trans-signaling is reliant upon the release of soluble IL6 receptor (sIL6R), which binds IL6 to create an agonistic IL6/sIL6R complex capable of activating cell types that would not normally respond to IL6 itself. Here we show that intrinsic and extrinsic apoptotic stimulation by DNA damage, cytokine deprivation, and Fas stimulation promotes shedding of sIL6R. Apoptosis-induced shedding of the IL6R was caspase dependent but PKC independent, with inhibition of ADAM17 preventing IL6R shedding. Such insight is relevant to the control of acute inflammation, where transition from the initial neutrophil infiltration to a more sustained population of mononuclear cells is essential for the resolution of the inflammatory process. This transitional event is governed by IL6 trans-signaling. This study demonstrates that IL6R is shed from apoptotic human neutrophils. In vivo studies in a murine inflammation model showed that neutrophil depletion resulted in reduced local sIL6R levels and a concomitant decrease in mononuclear cells, suggesting that apoptosis-induced IL6R shedding from neutrophils promotes IL6 trans-signaling and regulates the attraction of monocytic cells involved in the clearance of apoptotic neutrophils.

The heterodimeric cytokine IL-27 consists of the subunits p28 and EBI3. Hunter and colleagues demonstrate that p28 acting alone can inhibit the signaling of many cytokines by interfering with the common receptor gp130. The heterodimeric cytokine interleukin 27 (IL-27) signals through the IL-27Rα subunit of its receptor, combined with gp130, a common receptor chain used by several cytokines, including IL-6. Notably, the IL-27 subunits p28 (IL-27p28) and EBI3 are not always expressed together, which suggests that they may have unique functions. Here we show that IL-27p28, independently of EBI3, antagonized cytokine signaling through gp130 and IL-6-mediated production of IL-17 and IL-10. Similarly, the ability to generate antibody responses was dependent on the activity of gp130-signaling cytokines. Mice transgenic for expression of IL-27p28 showed a substantial defect in the formation of germinal centers and antibody production. Thus, IL-27p28, as a natural antagonist of gp130-mediated signaling, may be useful as a therapeutic for managing inflammation mediated by cytokines that signal through gp130.