DEL-1 is an endothelial cell-secreted protein that regulates LFA-1-integrin–dependent leukocyte recruitment and inflammation in various tissues. Here we identified a novel regulatory mechanism of DEL-1 in osteoclast biology. Specifically, we showed that DEL-1 is expressed by human and mouse osteoclasts and regulates their differentiation and resorptive function. Mechanistically, DEL-1 inhibited the expression of NFATc1, a master regulator of osteoclastogenesis, in a Mac-1-integrin–dependent manner. In vivo mechanistic analysis has dissociated the anti-inflammatory from the anti-bone resorptive action of DEL-1 and identified structural components thereof mediating these distinct functions. Importantly, locally administered human DEL-1 blocked inflammatory periodontal bone loss in nonhuman primates—a relevant model of human periodontitis. The ability of DEL-1 to regulate both upstream (inflammatory cell recruitment) and downstream (osteoclastogenesis) events that lead to inflammatory bone loss paves the way to a new class of endogenous therapeutics for treating periodontitis and perhaps other inflammatory disorders.

Cellular metabolism and signaling pathways are key regulators to determine conventional T cell fate and function, but little is understood about the role of cell metabolism for natural killer T (NKT) cell survival, proliferation, and function. We found that NKT cells operate distinct metabolic programming from CD4 T cells. NKT cells are less efficient in glucose uptake than CD4 T cells with or without activation. Gene-expression data revealed that, in NKT cells, glucose is preferentially metabolized by the pentose phosphate pathway and mitochondria, as opposed to being converted into lactate. In fact, glucose is essential for the effector functions of NKT cells and a high lactate environment is detrimental for NKT cell survival and proliferation. Increased glucose uptake and IFN-γ expression in NKT cells is inversely correlated with bacterial loads in response to bacterial infection, further supporting the significance of glucose metabolism for NKT cell function. We also found that promyelocytic leukemia zinc finger seemed to play a role in regulating NKT cells’ glucose metabolism. Overall, our study reveals that NKT cells use distinct arms of glucose metabolism for their survival and function.

Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is ϳ90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement.

The mammalian target of rapamycin (mTOR) senses and incorporates different environmental cues via the two signaling complexes mTORC1 and mTORC2. As a result, mTOR controls cell growth and survival and also shapes different effector functions of the cells including immune cells such as T cells. We demonstrate here that iNKT cell development is controlled by mTORC2 in a cell-intrinsic manner. In mice deficient in mTORC2 signaling due to the conditional deletion of the Rictor gene, iNKT cell numbers were reduced in the thymus and periphery. This is caused by decreased proliferation of stage 1 iNKT cells and poor development through subsequent stages. Functionally, iNKT cells devoid of mTORC2 signaling showed reduced number of IL-4-expressing cells, which correlated with a decrease in the transcription factor GATA-3-expressing cells. However, promyelocytic leukemia zinc-finger (PLZF), a critical transcription factor for iNKT cell development, is expressed at a similar level in mTORC2 deficient iNKT cells compared to that in the wild type iNKT cells. Furthermore, cellular localization of PLZF was not altered in the absence of mTOR2 signaling. Thus, our study reveals the PLZF-independent mechanisms of the development and function of iNKT cells regulated by mTORC2.

Variola and vaccinia viruses, the two most important members of the family Poxviridae, are known to encode homologs of the human complement regulators named smallpox inhibitor of complement enzymes (SPICE) and vaccinia virus complement control protein (VCP), respectively, to subvert the host complement system. Intriguingly, consistent with the host tropism of these viruses, SPICE has been shown to be more human complement-specific than VCP, and in this study we show that VCP is more bovine complement-specific than SPICE. Based on mutagenesis and mechanistic studies, we suggest that the major determinant for the switch in species selectivity of SPICE and VCP is the presence of oppositely charged residues in the central complement control modules, which help enhance their interaction with factor I and C3b, the proteolytically cleaved form of C3. Thus, our results provide a molecular basis for the species selectivity in poxviral complement regulators.

Developmental endothelial locus-1 (Del-1) is an endothelial cell-secreted protein that limits the recruitment of neutrophils by antagonizing the interaction between the LFA-1 integrin on neutrophils and the intercellular adhesion molecule (ICAM)-1 on endothelial cells. Mice with genetic or age-associated Del-1 deficiency exhibit increased neutrophil infiltration in the periodontium resulting in inflammatory bone loss. Here we investigated additional novel mechanisms whereby Del-1 could interfere with neutrophil recruitment and inflammation. Treatment of human endothelial cells with Del-1 did not affect the expression of endothelial molecules involved in the leukocyte adhesion cascade (ICAM-1, VCAM-1, and E-selectin). Moreover, genetic or age-associated Del-1 deficiency did not significantly alter the expression of these adhesion molecules in the murine periodontium, further ruling out altered adhesion molecule expression as a mechanism whereby Del-1 regulates leukocyte recruitment. Strikingly, Del-1 inhibited ICAM-1-dependent chemokine release (CXCL2, CCL3) by neutrophils. Therefore, Del-1 could potentially suppress the amplification of inflammatory cell recruitment mediated through chemokine release by infiltrating neutrophils. Interestingly, Del-1 was itself regulated by inflammatory stimuli, which generally exerted opposite effects on adhesion molecule expression. The reciprocal regulation between Del-1 and inflammation may contribute to optimally balance the protective and the potentially harmful effects of inflammatory cell recruitment.

Reactive oxygen species (ROS) are byproducts of aerobic metabolism and contribute to both physiological and pathological conditions as second messengers. ROS are essential for activation of T cells, but how ROS influence NKT cells is unknown. In the current study, we investigated the role of ROS in NKT cell function. We found that NKT cells, but not CD4 nor CD8 T cells, have dramatically high ROS in the spleen and liver of mice but not in thymus or adipose tissues. Accordingly, ROS-high NKT cells exhibited increased susceptibility and apoptotic cell death with oxidative stress. High ROS in the peripheral NKT cells were primarily produced by NADPH oxidases and not mitochondria. We observed that sorted ROS-high NKT cells were enriched in NKT1 and NKT17 cells, while NKT2 cells were dominant in ROS-low cells. Furthermore, treatment of NKT cells with antioxidants lead to reduced frequencies of IFN-γ– and IL-17-expressing cells, indicating that ROS play a role in regulating the inflammatory function of NKT cells. The transcription factor promyelocytic leukemia zinc finger (PLZF) seemed to control the ROS levels. NKT cells from adipose tissues that do not express PLZF and those from PLZF haplo-deficient mice have low ROS. Conversely, ROS were highly elevated in CD4 T cells from mice ectopically expressing PLZF. Thus our findings demonstrate that PLZF controls ROS levels, which in turn governs the inflammatory function of NKT cells.

Background & Aims The liver is an immunologically-privileged organ. Breakdown of liver immune privilege has been reported in chronic liver disease; however, the role of adaptive immunity in liver injury is poorly defined. NIK is known to regulate immune tissue development, but its role in maintaining liver homeostasis remains unknown. This study aimed to assess the role of NIK, particularly thymic NIK, in regulating liver adaptive immunity. Methods NIK was deleted systemically or conditionally using the cre/loxp system. CD4+ or CD8+ T cells were depleted using anti-CD4 or anti-CD8 antibody. Donor bone marrows or thymi were transferred into recipient mice. Immune cells were assessed by immunohistochemistry and flow cytometry. Results Global, but not liver-specific or hematopoietic lineage cell-specific, deletion of NIK induced fatal liver injury, inflammation, and fibrosis. Likewise, adoptive transfer of NIK-null, but not wild type, thymi into immune-deficient mice induced liver inflammation, injury, and fibrosis in recipients. Liver inflammation was characterized by a massive expansion of T cells, particularly the CD4+ T cell subpopulation. Depletion of CD4+, but not CD8+, T cells fully protected against liver injury, inflammation, and fibrosis in NIK-null mice. NIK deficiency also resulted in inflammation in the lung, kidney, and pancreas, but to a lesser degree relative to the liver. Conclusions Thymic NIK suppresses development of autoreactive T cells against liver antigens, and NIK deficiency in the thymus results in CD4+ T cell-orchestrated autoimmune hepatitis and liver fibrosis. Thus, thymic NIK is indispensable for the maintenance of liver immune privilege and liver homeostasis.