Objective: To investigate the clinical significance of lymphoid neogenesis (LN) in rheumatoid arthritis (RA), the clinicopathological correlates of this process and its evolution after anti-tumour necrosis factor (TNF)α therapy in a large series of synovial tissues were analysed. Methods: Arthroscopic synovial biopsies from 86 patients with RA were analysed by immunohistochemistry. LN was defined as the presence of large aggregates of lymphocytes with T/B cell compartmentalisation and peripheral node addressin (PNAd) positive high endothelial venules. Clinical variables at baseline and after prospective follow-up were compared in LN positive and negative RA subsets. The evolution of LN and its correlation with the clinical course in a subgroup of 24 patients that underwent a second arthroscopic biopsy after anti-TNFα therapy was also analysed. Results: LN was present in 49% of RA synovial tissues. Patients with LN had a significantly higher disease duration and a higher previous use of anti-TNFα agents. During prospective follow-up, the proportion of patients achieving good or moderate European League Against Rheumatism (EULAR) 28-joint Disease Activity Score (DAS28) responses was significantly lower in patients who were LN positive despite a significantly higher use of anti-TNFα agents. By multivariate logistic regression analysis, LN remained as an independent negative predictor of response to therapy. In the subgroup of patients rebiopsied after anti-TNFα therapy, reversal of LN features occurred in 56% of the patients and correlated with good clinical responses. Conclusions: Synovial LN in RA predicts a lower response to therapy. LN features can be reversed after a short period of anti-TNFα therapy in parallel to good clinical responses.

Macrophages occupy a prominent position during immune responses. They are considered the final effectors of any given immune response since they can be activated by a wide range of surface ligands and cytokines to acquire a continuum of functional states. Macrophages are involved in tissue homeostasis and in the promotion or resolution of inflammatory responses, causing tissue damage or helping in tissue repair. Knowledge in macrophage polarization has significantly increased in the last decade. Biomarkers, functions, and metabolic states associated with macrophage polarization status have been defined both in murine and human models. Moreover, a large body of evidence demonstrated that macrophage status is a dynamic process that can be modified. Macrophages orchestrate virtually all major diseases—sepsis, infection, chronic inflammatory diseases (rheumatoid arthritis), neurodegenerative disease, and cancer—and thus they represent attractive therapeutic targets. In fact, the possibility to “reprogram” macrophage status is considered as a promising strategy for designing novel therapies. Here, we will review the role of different tissue macrophage populations in the instauration and progression of inflammatory and non-inflammatory pathologies, as exemplified by rheumatoid arthritis, osteoporosis, glioblastoma, and tumor metastasis. We will analyze: 1) the potential as therapeutic targets of recently described macrophage populations, such as osteomacs, reported to play an important role in bone formation and homeostasis or metastasis-associated macrophages (MAMs), key players in the generation of premetastatic niche; 2) the current and potential future approaches to target monocytes/macrophages and their inflammation-causing products in rheumatoid arthritis; and 3) the development of novel intervention strategies using oncolytic viruses, immunomodulatory agents, and checkpoint inhibitors aiming to boost M1-associated anti-tumor immunity. In this review, we will focus on the potential of macrophages as therapeutic targets and discuss their involvement in state-of-the-art strategies to modulate prevalent pathologies of aging societies.

Macrophages integrate information from the tissue microenvironment and adjust their effector functions according to the prevalent extracellular stimuli. Therefore, macrophages can acquire a variety of activation (polarization) states, and this functional plasticity allows the adequate initiation, regulation, and resolution of inflammatory responses. Modulation of the glucose metabolism contributes to the macrophage adaptation to the surrounding cytokine milieu, as exemplified by the distinct glucose catabolism of macrophages exposed to LPS/IFN-γ or IL-4. To dissect the acquisition of macrophage effector functions in the absence of activating cytokines, we assessed the bioenergetic profile of macrophages generated in the presence of GM-CSF (GM-MØ) or M-CSF (M-MØ), which do not release pro- or anti-inflammatory cytokines unless subjected to additional activating stimuli. Compared to M-MØ, GM-MØ displayed higher oxygen consumption rate and aerobic glycolysis (extracellular acidification rate [ECAR]), as well as higher expression of genes encoding glycolytic enzymes. However, M-MØ exhibited a significantly higher oxygen consumption rate/ECAR ratio. Surprisingly, whereas aerobic glycolysis positively regulated IL1B, TNF, and INHBA mRNA expression in both macrophage subtypes, mitochondrial respiration negatively affected IL6, IL1B, TNF, and CXCL10 mRNA expression in M-MØ. The physiological significance of these results became evident under low oxygen tensions, as hypoxia enhanced ECAR in M-MØ via HIF-1α and HIF-2α, increased expression of glycolytic enzymes and GM-MØ–specific genes, and diminished expression of M-MØ–associated genes. Therefore, our data indicate that GM-MØ and M-MØ display distinct bioenergetic profiles, and that hypoxia triggers a transcriptomic switch in macrophages by promoting a HIF-1α/HIF-2α-dependent increase in ECAR.

Objective Hyperplasia and phenotypic changes in fibroblasts are often observed in chronic inflammatory lesions, and yet the autonomous pathogenic contribution of these changes is uncertain. The purpose of this study was to analyze the intrinsic ability of fibroblasts from chronically inflamed synovial tissue to drive cell recruitment and angiogenesis. Methods Fibroblasts from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), as well as fibroblasts from healthy synovial tissue and healthy skin, were cultured and subcutaneously engrafted into immunodeficient mice. Cell infiltration and angiogenesis were analyzed in the grafts by immunohistochemical studies. The role of vascular endothelial growth factor (VEGF), CXCL12, and hypoxia-inducible transcription factor 1α (HIF-1α) in these processes was investigated using specific antagonists or small interfering RNA (siRNA)–mediated down-regulation of HIF-1α in fibroblasts. Results Inflammatory (OA and RA) synovial fibroblasts, compared with healthy dermal or synovial tissue fibroblasts, induced a significant enhancement in myeloid cell infiltration and angiogenesis in immunodeficient mice. These activities were associated with increased constitutive and hypoxia-induced expression of VEGF, but not CXCL12, in inflammatory fibroblasts compared with healthy fibroblasts. VEGF and CXCL12 antagonists significantly reduced myeloid cell infiltration and angiogenesis. Furthermore, targeting of HIF-1α expression by siRNA or of HIF-1α transcriptional activity by the small molecule chetomin in RA fibroblasts significantly reduced both responses. Conclusion These results demonstrate that chronic synovial inflammation is associated with stable fibroblast changes that, under hypoxic conditions, are sufficient to induce inflammatory cell recruitment and angiogenesis, both of which are processes relevant to the perpetuation of chronic inflammation.

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12γ is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12γ through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12γ both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12γ strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12γ one of the higher affinity for HS (K_d = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12γ to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12α. In good agreement, mutant CXCL12γ chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12γ features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12γ the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.