Ma et al. demonstrated that R3+ macrophages are involved in the active elimination of Salmonella typhimurium via AMPK-mediated glycolytic reprogramming and inflammasome activation. Upon infection, macrophages expressed FLT4/VEGFR-3 and its ligand VEGF-C, and this signaling was involved in the inhibition of CASP1 (caspase 1)-dependent inflammasome activation and pyroptosis; it also enhanced MAP1LC3/LC3 activation for bacterial elimination (58). Zhang and colleagues utilized macrophage-specific VEGFR-3 knockout mice (Vegfr-3 ^fl/flLyzM-Cre^+/−) to demonstrate that activated R3+ macrophage prevents endotoxin shock by limiting Toll-like receptor-4 (TLR-4)/NF-κB signaling and thereby represented a self-control mechanism during antibacterial innate immunity. Gram-negative bacterial infection activated VEGF-C/VEGFR-3 signaling in macrophages and enhanced SOCS1 (suppressor of cytokine signaling 1) expression to inhibit the TLR-4/NF-κB pathway (28). Vegfr-3 ^fL/flLyzM-Cre^+/− mice were also used to demonstrate that R3+ macrophages restrain neuronal damage post-encephalitis by limiting TNF-α production. Brain macrophages from Vegfr-3 ^fl/flLyzM-Cre^+/− mice infected with Japanese encephalitis virus (JEV) were sequenced to identify some of the top activated signaling pathways that were associated with inflammation and infection including the PI3K-AKT pathway. Peritoneal exudate macrophages (PEMS) from these mice showed reduced levels of p-AKT1 after treatment with JEV-infected N2a (Neuro2a cells). VEGF-C treatment had an inhibitory effect on TNF-α production in wild-type PEMS that was restored by uprosertib, an AKT inhibitor. This suggests that macrophage VEGFR-3 signaling and the activation of the PI3K-AKT pathway inhibits TNF-α secretion in the brain after neurotropic viral infection (40). Taken together, R3+ macrophages appear to exhibit a positive response to infections.

In a model of experimental colitis, mice injected with carboxylated crimson, fluorescent LPS-coated beads showed increased clearance of bead+ macrophages from the inflamed colon with VEGF-C treatment (27). Macrophages isolated from colitic mice showed a significant increase in phosphorylated STAT6 upon VEGF-C treatment with the effect being inhibited by an anti-VEGFR-3 antibody. Inhibition of STAT6 in VEGF-C-treated cells abolished the ability of VEGF-C to alter “M2” and “M1” macrophage-associated genes as well as macrophage migration. In vitro, VEGF-C-induced bone marrow-derived macrophages ameliorated clinical parameters in the experimental colitic mice, whereas this protective role was abolished by STAT6-silenced VEGF-C-induced macrophages. This suggests that VEGF-C/VEGFR-3 signaling in macrophages provided protection against the development of acute and chronic colitis by increasing cell mobilization and bacterial antigen clearance from the inflamed colon to draining lymph nodes (27).

R3+ macrophages contributed to prolonged survival and decreased the degree of lung injury in a sepsis model through reducing TLR-4 signaling and pro-inflammatory cytokines (28). A study by Yamashita et al. (59) demonstrated that R3+ macrophages ameliorated experimental lung injury through a series of gain and loss of VEGFC/VEGFR-3 signaling function experiments that employed adenovirus-mediated intranasal delivery of VEGF-C (Ad-VEGF-C vector) and soluble VEGFR-3 or anti-VEGFR-3 blocking antibodies. They also utilized LysM-specific VEGFR-3 knockout mice produced from LysM-Cre and VEGFR-3-floxed mice and reported that VEGF-C/VEGFR-3 signaling increased efferocytosis via upregulation of integrin α_v in macrophages. In addition, they showed a significant decrease in VEGFR-3 expression and efferocytosis in human monocyte-derived macrophages upon incubation with bronchoalveolar lavage fluid from acute respiratory distress syndrome (ARDS) patients, suggesting that the protective function of macrophages is potentially impaired in patients during ARDS due to decreased levels of R3+ macrophages.

Maruyama and colleagues identified that high-glucose conditions caused a reduction in VEGFR-3 expression in macrophages that could be rescued by stimulation with IL-1β. Following rescue, application of these R3+ macrophages to wounds in diabetic mice induced lymphangiogenesis and accelerated wound healing. The authors posited that these macrophages play a direct role in vessel formation. This suggests that the decrease in R3+ macrophages in diabetic mice may have contributed to a reduction in lymphatic vessels resulting in impaired dermal wound healing (42).

Dermal immunology studies carried out in mice expressing soluble VEGFR-3 (a VEGF-C/D trap) in the skin identified fewer macrophages in the dermal-draining lymph nodes compared to normal mice (60). These mice demonstrated reduced immune tolerization, even developing an autoimmune response to their skin. Increased levels of VEGF-C/D are associated with inflammation and cancer progression and play a critical role in lymphatic and lymph node structure remodeling (61–63). Whether macrophage VEGFR-3 signaling could also play a role in immune response propagation through lymph node sinus remodeling or lymphatic–immune interactions specifically in this mutant model or generally in other models remains unknown. For example, subcapsular sinus macrophages in lymph nodes prevent the systemic spread of lymph-borne pathogens and initiate humoral responses (64). Inhibition of VEGFR-3 signaling reduced not only lymph node lymphatic proliferation and inflammation, but also the expression of IL-1β in subcapsular sinus macrophages following stroke (65). What role macrophage VEGFR-3 signaling plays in lymph node lymphangiogenesis or how VEGFR-3-mediated remodeling impacts polarization during inflammation thus remains unclear.

A study utilizing a rat model of orthotopic liver transplantation demonstrated a protective role of VEGFC/VEGFR-3 signaling on ischemic reperfusion injury by modifying macrophage polarization from the so-called pro-inflammatory “M1” to beneficial “M2” phenotype. Continuous perfusion of donor livers with VEGF-C prior to transplantation significantly reduced post-operative serum alanine transaminase (ALT) and total bilirubin (TBIL) level and also suppressed the NF-κB activity and upregulated the expression of SOCS1 and p-GSK3β in Kupffer cells and shifted the M1/M2 balance towards an anti-inflammatory profile (66). In another mouse study involving hepatic ischemia–reperfusion injury, treatment with recombinant VEGF-D facilitated liver repair with expansion of lymphatic vessels and increased expression of R3+ “reparative” macrophages (defined by the authors as Ly6C^low/CD11b^high/F4/80^high/CX3CR1^high/CCR2^low). Conversely, VEGFR-3 inhibition suppressed liver repair, drainage function, and accumulation of R3+ reparative macrophages, suggesting that activation of VEGFR-3 signaling induced lymphangiogenesis and increased the number of reparative macrophages essential for liver repair (29).

Zhang et al. (47) utilized unilateral ureteral obstruction (UUO) and the adriamycin nephropathy mouse model (ARD) to demonstrate the association between renal fibrosis-associated lymphangiogenesis and VEGFR-3-dependent macrophage polarization. VEGF-C-treated macrophages formed tube-like structures in Matrigel in vitro and, similarly, adoptive transfusion of primary M1 macrophages into the UUO and ARD mice formed tube-like structures in the fibrotic kidneys. They also identified that the induction of autophagy in macrophages by rapamycin decreased M1 macrophage polarization and differentiation into LECs whereas activation of the VEGF-C/VEGFR-3 pathway downregulated macrophage autophagy and subsequently promoted M1 macrophage polarization. These results suggested that M1 macrophages promoted lymphangiogenesis and contributed to newly formed lymphatic vessels in the renal fibrosis microenvironment, and that VEGF-C/VEGFR3 signaling promoted macrophage M1 polarization by suppressing macrophage autophagy and then increased the transdifferentiation of M1 macrophages into LECs. Another study using db/db mice demonstrated that VEGFR-3 activation increases lymphatic proliferation and M1 macrophage infiltration during tubulointerstitial fibrosis induced by diabetic nephropathy. In this study, palmitate-induced lipotoxicity enhanced M1 phenotypes in RAW264.7 monocytes/macrophages and increased VEGFR-3 and Lyve-1 expression, which were inhibited by SAR131675 (VEGFR-3 inhibitor) treatment, suggesting that inhibiting VEGFR-3 signaling can prevent diabetic nephropathy by inhibiting renal lymphangiogenesis mediated through macrophage polarization under lipotoxic conditions (67).

R3+ macrophages also play a significant role in tumor-associated lymphangiogenesis and cancer cell metastasis. The skin surrounding VEGF-C-transfected melanomas from mice showed a markedly increased density of R3+ macrophages. In vitro studies from mouse peritoneal macrophages revealed VEGF-C-induced chemotaxis of macrophages in a dose-dependent manner (33). Macrophages from chemotherapy-treated mice induced lymphangiogenesis in a VEGFR-3-dependent manner by promoting the secretion of cathepsins, leading to the upregulation of VEGF-C by heparanase activity. Blocking VEGFR-3+ tumor-associated macrophages (TAMs) inhibited lymphangiogenesis and subsequent metastasis (48). VEGFR-3+ TAMs increased the VEGF-C/D expression in the bladder tumor mass, resulting in increased lymphatic metastases (39). VEGFC-dependent activation of VEGFR-3 in colorectal TAMs weakened antitumor adaptive immunity promoting cancer immune escape; these effects were abolished upon VEGFR-3 inhibition (68). VEGFR-3-expressing TAMs are also involved in the production of VEGF-C and VEGF-D within the peritumoral stroma, resulting in peritumoral lymphangiogenesis in human cancer (36). Dalton and colleagues identified that R3+ macrophages in the tumor microenvironment orchestrate anti-VEGF therapy resistance. Biopsy specimens from ovarian cancer patients who were resistant to anti-VEGF therapy showed reduced macrophage VEGFR-3 expression. Anti-VEGF antibody (AVA)-resistant immortalized murine macrophages secreted less VEGF and showed increased secretion of alternative pro-angiogenic cytokines and chemokines. These macrophages revealed a significant increase in the methylation at the VEGFR-3 promoter region, suggesting that at the emergence of resistance, depletion of VEGFR-3 expression in macrophages is accompanied by an upregulation of alternative angiogenic pathways, thus facilitating escape from anti-VEGF therapy (69). VEGFR-3 tyrosine kinase activity is also inhibited by VEGFR-targeting and less specific tyrosine kinase inhibitors in use or in development for cancer anti-angiogenic or anti-metastatic effects, such as sunitinib, fruquintinib, or cediranib. Whether reduced vascularization and macrophage numbers, or changes in macrophage polarization, reported with their use are directly through VEGFR-3 activity on macrophages is unclear (26, 70, 71).

Karaman and colleagues demonstrated that subcutaneous white adipose tissue (SWAT) of high-fat diet (HFD)-fed K14-VEGFR-3-Ig (sR3) mice that constitutively express soluble-VEGFR-3-Ig in the skin, scavenging VEGF-C and -D, had an increased M2/M1 marker expression ratio. A transwell migration experiment revealed VEGFC/D to be highly chemotactic to adipose tissue macrophages particularly M1 macrophages that express heightened VEGFR-3. Antibody mediated depletion of VEGFR-3 in db/db mice reduced macrophage infiltration in adipose tissue, reduced adipose tissue inflammation, and reduced obesity-induced insulin resistance. They suggested that VEGF-C/D plays an important role in recruiting the M1-polarized R3+ macrophages in obese adipose tissue and in the development of obesity-associated insulin resistance (50). Another study from the same group using K14-VEGF-C mice that overexpress human VEGF-C under control of the keratin-14 promoter in epidermal keratinocytes revealed the recruitment of R3+ inflammatory macrophages into SWAT prior to the emergence of weight gain that helped in promoting insulin resistance (51). Overexpression of VEGF-D in adipose tissue of lean mice also drove adipose macrophage infiltration, inflammation, and fibrosis (72).