The coordinated behavior of proteins is central to systems biology. However, the underlying mechanisms are poorly known and methods to analyze coordination by conventional quantitative proteomics are still lacking. We present the Systems Biology Triangle (SBT), a new algorithm that allows the study of protein coordination by pairwise quantitative proteomics. The Systems Biology Triangle detected statistically significant coordination in diverse biological models of very different nature and subjected to different kinds of perturbations. The Systems Biology Triangle also revealed with unprecedented molecular detail an array of coordinated, early protein responses in vascular smooth muscle cells treated at different times with angiotensin-II. These responses included activation of protein synthesis, folding, turnover, and muscle contraction – consistent with a differentiated phenotype—as well as the induction of migration and the repression of cell proliferation and secretion. Remarkably, the majority of the altered functional categories were protein complexes, interaction networks, or metabolic pathways. These changes could not be detected by other algorithms widely used by the proteomics community, and the vast majority of proteins involved have not been described before to be regulated by AngII. The unique capabilities of The Systems Biology Triangle to detect functional protein alterations produced by the coordinated action of proteins in pairwise quantitative proteomics experiments make this algorithm an attractive choice for the biological interpretation of results on a routine basis.

The interferon (IFN)-stimulated gene 15 (ISG15) encodes one of the most abundant proteins induced by interferon, and its expression is associated with antiviral immunity. To identify protein components implicated in IFN and ISG15 signaling, we compared the proteomes of ISG15^-/- and ISG15^+/+ bone marrow derived macrophages (BMDM) after vaccinia virus (VACV) infection. The results of this analysis revealed that mitochondrial dysfunction and oxidative phosphorylation (OXPHOS) were pathways altered in ISG15^-/- BMDM treated with IFN. Mitochondrial respiration, Adenosine triphosphate (ATP) and reactive oxygen species (ROS) production was higher in ISG15^+/+ BMDM than in ISG15^-/- BMDM following IFN treatment, indicating the involvement of ISG15-dependent mechanisms. An additional consequence of ISG15 depletion was a significant change in macrophage polarization. Although infected ISG15^-/- macrophages showed a robust proinflammatory cytokine expression pattern typical of an M1 phenotype, a clear blockade of nitric oxide (NO) production and arginase-1 activation was detected. Accordingly, following IFN treatment, NO release was higher in ISG15^+/+ macrophages than in ISG15^-/- macrophages concomitant with a decrease in viral titer. Thus, ISG15^-/- macrophages were permissive for VACV replication following IFN treatment. In conclusion, our results demonstrate that ISG15 governs the dynamic functionality of mitochondria, specifically, OXPHOS and mitophagy, broadening its physiological role as an antiviral agent. Protein modification by ubiquitin and ubiquitin-like proteins is a key regulatory process of the innate and adaptive immune response. Interferon-stimulated gene 15 product (ISG15) is an ubiquitin-like protein modifier that can reversibly attach to different viral and cellular proteins, mediating potent antiviral responses. In turn, many viruses, including poxviruses, have evolved strategies to antagonize the antiviral and inflammatory effects of the innate immune response in order to keep infected cells alive until virus replication is complete. Here, we describe a novel role for ISG15 in the control of mitochondrial function. Post-translational modifications such as ISGylation regulate essential mitochondrial processes including respiration and mitophagy, and influence macrophage innate immunity signaling. These findings are clinically relevant since mitochondrial dysfunction is seen in many pathologies, such as infectious disease, cancer, and cardiovascular or neurological disorders, among others, underscoring the importance of the relationship between cellular metabolism and immune response.

Recent evidence on HDAC6 function underlines its role as a key protein in the innate immune response to viral infection. However, whether HDAC6 regulates innate immunity during bacterial infection remains unexplored. To assess the role of HDAC6 in the regulation of defence mechanisms against intracellular bacteria, we used the Listeria monocytogenes (Lm) infection model. Our data show that Hdac6^-/- bone marrow-derived dendritic cells (BMDCs) have a higher bacterial load than Hdac6^+/+ cells, correlating with weaker induction of IFN-related genes, pro-inflammatory cytokines and nitrite production after bacterial infection. Hdac6^-/- BMDCs have a weakened phosphorylation of MAPK signalling in response to Lm infection, suggesting altered Toll-like receptor signalling (TLR). Compared with Hdac6^+/+ counterparts, Hdac6^-/- GM-CSF-derived and FLT3L-derived dendritic cells show weaker pro-inflammatory cytokine secretion in response to various TLR agonists. Moreover, HDAC6 associates with the TLR-adaptor molecule Myeloid differentiation primary response gene 88 (MyD88), and the absence of HDAC6 seems to diminish the NF-κB induction after TLR stimuli. Hdac6^-/- mice display low serum levels of inflammatory cytokine IL-6 and correspondingly an increased survival to a systemic infection with Lm. The impaired bacterial clearance in the absence of HDAC6 appears to be caused by a defect in autophagy. Hence, Hdac6^-/- BMDCs accumulate higher levels of the autophagy marker p62 and show defective phagosome-lysosome fusion. These data underline the important function of HDAC6 in dendritic cells not only in bacterial autophagy, but also in the proper activation of TLR signalling. These results thus demonstrate an important regulatory role for HDAC6 in the innate immune response to intracellular bacterial infection. Listeria monocytogenes (Lm) is a food-borne intracellular bacterium that causes listeriosis to 1.600 people each year, being responsible of approximately 260 deaths. This pathogen mostly affects immunocompromised individuals and pregnant women. It is particularly dangerous for the later due to its ability to pass across the placenta and the blood-brain barrier. Lm is extensively used as a Gram positive infection model in the laboratory to study innate and adaptive immune responses. HDAC6 is an important regulatory enzyme of the tubulin and actin cytoskeletons. Its inhibition or deficiency quells the immune response against different virus infections. Previous work has shown its involvement in the regulation of viral RNA-sensing activity and in interferon signalling. In this study, we report that HDAC6 is an essential component of the innate immune response to fight against intracellular bacterial infections. Genetic ablation of HDAC6 impairs activation of the pertinent Toll-like receptor pathway to induce the pro-inflammatory transcriptional program of the cell. Moreover, this enzyme controls cytoskeletal proteins that mediate the fusion of phagosome-contained bacteria with the lysosome during pathogen degradation.

Borreliae employ multiple immune evasive strategies such as binding to complement regulatory proteins [factor H (fH) and factor H like-1 (FHL1)], differential regulation of surface membrane proteins, antigenic variation, and binding of plasminogen/plasmin and matrix metalloproteinases. As a complement regulatory subunit, fH serves as a cofactor for the factor I-mediated cleavage of C3b. fH binding by Borrelia has been correlated with pathogenesis as well as with host diversity. Here we show the differential binding of borrelial proteins to fH from human and animal sera.

Matrix metalloproteinases are involved in vascular remodeling. Little is known about their immune regulatory role in atherosclerosis. Here we show that mice deficient for MT4-MMP have increased adherence of macrophages to inflamed peritonea, and larger lipid deposits and macrophage burden in atherosclerotic plaques. We also demonstrate that MT4-MMP deficiency results in higher numbers of patrolling monocytes crawling and adhered to inflamed endothelia, and the accumulation of Mafb+ apoptosis inhibitor of macrophage (AIM)+ macrophages at incipient atherosclerotic lesions in mice. Functionally, MT4-MMP-null Mafb+AIM+ peritoneal macrophages express higher AIM and scavenger receptor CD36, are more resistant to apoptosis, and bind acLDL avidly, all of which contribute to atherosclerosis. CCR5 inhibition alleviates these effects by hindering the enhanced recruitment of MT4-MMP-null patrolling monocytes to early atherosclerotic lesions, thus blocking Mafb+AIM+ macrophage accumulation and atherosclerosis acceleration. Our results suggest that MT4-MMP targeting may constitute a novel strategy to boost patrolling monocyte activity in early inflammation.