Ovariectomized <i>Ncf1</i> knockout females developed severe CIA similar to the knockout male littermates.

<p>The prevalence of paw inflammation is presented in A, and the severity of the disease in B. The severity of the disease after exclusion of the animals that never developed any symptoms is shown in C. Anti-collagen antibodies in the serum during CIA reflects disease severity (D). The uteri were weighed after euthanasia to verify successful removal of the ovaries (E). m = males, f = females, OVX = ovariectomized. Statistical significances between the genders in (D) were calculated by unpaired Student’s t-test (*<i>P</i><0.05). The grey box (in A–C) highlights the disease phase between days 30 and 50 post immunization. The data is presented as mean +/-SEM in B-D and n = 8–11 in A-B and D, n = 7 in C.</p

<i>Ncf1</i> knockouts and <i>Ncf1</i>^<i>m1J</i> mice are deficient in ROS production and share the interferon signature.

<p>(A) Intracellular ROS production is absent in blood granulocytes from <i>Ncf1</i>-deficient mice. Blood leukocytes from <i>Ncf1</i> knockouts (KO), wild types (WT) and mice with the <i>Ncf1</i>^<i>m1J</i> mutation were stained for surface markers and stimulated by PMA. Intracellular ROS production was quantified as dihydrorhodamine-123 fluorescence by flow cytometry. F = females, M = males. (B) <i>Ncf1</i> knockouts do not express the NCF1 protein in blood granulocytes (Gr-1+) when analyzed by intracellular staining and flow cytometry. T cells (CD3+CD8+) were used as negative control. (C) <i>Ncf1</i> knockouts fail to respond to PMA stimulation by extracellular ROS production. Bone marrow (BM), blood (WBC for white blood cells) and spleen (SPL) cell suspensions were depleted of erythrocytes and stimulated in the presence of isoluminol, a cell impermeable chemiluminescent substrate. Extracellular ROS production is presented as signal after 30 min stimulation. RLU = relative luminescence units. (D) STAT1 expression is upregulated in the absence of functional <i>Ncf1</i>. Blood cells from naïve mice were stained for cell surface markers. STAT1 expression was analyzed by intracellular staining, quantified by flow cytometry, and analyzed in granulocytic (Gr-1+) monocytic (CD11b+ Gr-1 negative) cells and B cells (B220+). The WT group comprises of mice with the wild type <i>Ncf1</i> either as homozygous or heterozygous (+/KO). Statistical significances between the female genotypes were calculated with unpaired Student’s t-test and presented as <i>P</i>-value or indicated as * <i>P</i><0.05 and ** <i>P</i><0.01. The data is presented as a box-and-whisker blot showing the quartiles (A) or as mean +/-SEM (B-D). Graphs (A), (SPL in C) and (D) present pooled data from three, two or four independent experiments, respectively. Number of mice per genotype: n = 6–12 in A; n = 4–5 in B; n = 4–15 in C; n = 6–13 in D. MFI = mean fluorescence intensity.</p

Methylcholanthrene-Induced Sarcomas Develop Independently from NOX2-Derived ROS

<div><p>Reactive oxygen species (ROS) produced by the inducible NADPH oxidase type 2 (NOX2) complex are essential for clearing certain infectious organisms but may also have a role in regulating inflammation and immune response. For example, ROS is involved in myeloid derived suppressor cell (MDSC)- and regulatory T cell (T_reg) mediated T- and NK-cell suppression. However, abundant ROS produced within the tumor microenvironment, or by the tumor itself may also yield oxidative stress, which can blunt anti-tumor immune responses as well as eventually leading to tumor toxicity. In this study we aimed to decipher the role of NOX2-derived ROS in a chemically (by methylcholanthrene (MCA)) induced sarcoma model. Superoxide production by NOX2 requires the p47^phox (NCF1) subunit to organize the formation of the NOX2 complex on the cell membrane. Homozygous mutant mice (NCF1*^/*) have a functional loss of their super oxide burst while heterozygous mice (NCF1*^/+) retain this key function. Mice harboring either a homo- or a heterozygous mutation were injected intramuscularly with MCA to induce sarcoma formation. We found that NOX2 functionality does not determine tumor incidence in the tested MCA model. Comprehensive immune monitoring in tumor bearing mice showed that infiltrating immune cells experienced an increase in their oxidative state regardless of the NOX2 functionality. While MCA-induced sarcomas where characterized by a T_reg and MDSC accumulation, no significant differences could be found between NCF1*^/* and NCF1*^/+ mice. Furthermore, infiltrating T cells showed an increase in effector-memory cell phenotype markers in both NCF1*^/* and NCF1*^/+ mice. Tumors established from both NCF1*^/* and NCF1*^/+ mice were tested for their <i>in vitro</i> proliferative capacity as well as their resistance to cisplatin and radiation therapy, with no differences being recorded. Overall our findings indicate that NOX2 activity does not play a key role in tumor development or immune cell infiltration in the chemically induced MCA sarcoma model.</p></div

Growth of MCA induced tumors is not affected by NCF1 mutation.

<p>(A) NCF1*^/* (n = 6) and NCF1*^/+ (n = 10) mice were tested for their ability to generate superoxide through stimulation of NOX2 with PMA. (B) NCF1*^/* and NCF1*^/+ mice (n = 54, 27 per group) were injected with 125 μg MCA dissolved in 25 μl of corn oil. Tumor incidence was followed through palpation of the injected muscle site. (C) Tumors were resected from MCA injected NCF1*^/* (n = 6) and NCF1*^/+ (n = 6) mice and passaged once in WT C57Bl/6 mice prior to transplantation of 5³ mm tumor into four WT C57Bl/6 (n = 48) mice in which (Ci) survival (Cii) and tumor growth were monitored.</p

MCA induced tumors accumulate immune regulatory cells.

<p><b>(</b>A) MDSCs as characterized by CD11b+ and GR1+ were gated from splenocytes or tumor samples as a percentage of live cells. (B) Percentage of live splenocytes that are MDSCs in tumor bearing NCF1*^/* and NCF1*^/+ mice compared to WT mice. (C) Percentage of live cells which are MDSC in tumors from both NCF1*^/* and NCF1*^/+ mice. (D) Percent FoxP3+ cells of CD4+ cells in spleens of tumor bearing mice compared to WT mice. (E) Percent T_reg cells in the blood, spleen and tumor of MCA tumor bearing mice.</p

Increased Levels of ROS Alter Cell Distributions

<div><p>(A) The phytol-induced increase in oxidative burst in granulocytes is not organ specific. Granulocytes taken from blood, LNs, and spleen on day 5 after phytol injection were analyzed for oxidative burst with and without PMA stimulation (<i>n</i> = 5).</p> <p>(B) Phytol increases oxidative burst in T cells with or without PMA stimulation 5 d after injection (<i>n</i> = 5). (A) and (B) are taken from different experiments and levels should thus not be compared.</p> <p>(C) ROS production in T cells is low compared to that in granulocytes. ROS production with and without PMA stimulation in spleen granulocytes and T cells from a naïve DA.<i>Ncf1</i>^DA rat.</p> <p>(D) Cell distribution is altered after phytol. Cell distribution in different organs analyzed 5 d after phytol by staining for B and T cells (<i>n</i> = 5).</p> <p>(E) The phytol effect is not mediated by a general immune-suppressive effect. Total levels of IgG and IgM antibodies in plasma measured 5 d after phytol injection expressed as relative values (<i>n</i> = 5).</p> <p>(F) Phytol does not increase cell death. Percentage of apoptotic (Annexin V+ and PI−) and necrotic (Annexin V+ and PI+) CD4⁺ cells ex vivo 5 d after phytol injection (<i>n</i> = 5).</p> <p>*<i>p</i> < 0.05, **<i>p</i> < 0.01.</p></div

Phytol Suppresses Type II Collagen Autoimmunity

<div><p>(A) Phytol prevents collagen-induced arthritis in DA.<i>Ncf1</i>^DA rats. Effect of SC administration of phytol (200 μl) 5 d before immunization with CII (<i>n</i> = 7).</p> <p>(B) Phytol decreases anti-CII antibody levels in plasma. At day 27 after immunization, plasma samples were taken and analyzed for anti-CII IgG and IgM levels (<i>n</i> = 7). Titres are relative to pooled positive plasma (units).</p> <p>(C) Phytol does not decrease total antibody levels. Total levels of IgG and IgM in plasma taken day 27 after immunization compared to pooled positive plasma (<i>n</i> = 7).</p> <p>(D) Phytol reduces arthritis severity also in NOCIA, an arthritis model induced without involvement of oil. Preventive effect on arthritis of phytol injection (200 μl SC) 5 d prior to induction of NOCIA in DA.<i>Ncf1</i>^DA rats (control <i>n</i> = 7; phytol <i>n</i> = 8).</p> <p>(E) DTH response is abolished after phytol injection. Day 67 after NOCIA immunization the immune system was challenged by an injection of CII in the ear. Subsequent increase in ear volume was measured and compared to control ear (control <i>n</i> = 7; phytol <i>n</i> = 8).</p> <p>*<i>p</i> < 0.05, **<i>p</i> < 0.01.</p></div

Phytol Increases Oxidative Bursts In Vivo

<div><p>(A) Phytol (Ph) restores the oxidative burst in vivo. Oxidative burst response to PMA stimulation in vitro in spleen granulocytes taken 5 d after SC phytol injection in DA.<i>Ncf1</i>^DA rats (200 μl) or from naïve DA.<i>Ncf1</i>^E3 compared to naïve DA.<i>Ncf1</i>^DA (<i>n</i> = 5).</p> <p>(B) Increased levels of products from lipid peroxidation in serum after phytol injection. Sera were taken 5 d after SC phytol injection in DA.<i>Ncf1</i>^DA and analyzed for levels of malondialdehyde, which reflects lipid peroxidation (<i>n</i> = 4).</p> <p>(C) Phytol injection has a long-term effect on oxidative burst capacity of granulocytes. Capacity of DA.<i>Ncf1</i>^DA blood granulocytes to exert an oxidative burst in response to PMA stimulation in vitro after SC injection of phytol or pristane (200 μl) on day 0. Values are presented as percent of oxidative burst compared naïve DA.<i>Ncf1</i>^DA rats (represented by the line) (<i>n</i> = 5).</p> <p>(D) Phytol increases the oxidative burst capacity in BM granulocytes. Oxidative burst capacity of granulocytes from BM taken 3 hours (<i>n</i> = 5) and 24 hours (<i>n</i> = 4) after SC phytol (Ph; 200 μl) injection in DA.<i>Ncf1</i>^DA rats or from naïve DA.<i>Ncf1</i>^E3, compared to naive DA.<i>Ncf1</i>^DA. ^#Experiments were performed independent of each other and levels should thus not be compared.</p> <p>*<i>p</i> < 0.05, **<i>p</i> < 0.01.</p></div

Phytol Suppresses Established Arthritis

<div><p>(A) Phytol injection ameliorates acute arthritis. Therapeutic effect of phytol in acute phase of PIA in DA.<i>Ncf1</i>^DA rats after two injections (200 μl) 5 d apart (arrows) starting at the estimated peak of disease (day 22). (Significance indications above line represent SC administration and below IP administration) (control <i>n</i> = 18, IP <i>n</i> = 17, and SC <i>n</i> = 16).</p> <p>(B) Cartilage destruction is decreased after phytol injection. Plasma were taken day 38 and analyzed for COMP levels as a measurement of cartilage destruction. Results are presented as circles, where each circle represents one individual and filled circles represent rats with a mean reduction in score after day of first treatment (day 22). Lines represent mean values.</p> <p>(C) Phytol decreases arthritis severity in the chronic phase of arthritis. Phytol was injected IP twice (200 μl) (arrows), starting day 70 after pristane injection in DA.<i>Ncf1</i>^DA rats with 3 d between treatments (control, <i>n</i> = 19; phytol, <i>n</i> = 18).</p> <p>(D) Phytol affects macroscopic and microscopic inflammation. Pristane-injected rats treated with phytol on either day 0 or day 13 (<i>n</i> = 4). On day 22 rats were sacrificed and paws taken for histology and stained with erythrosine-hematoxylin (photomicrographs to right of graph; 20× magnification).</p> <p>(E) The preventive effect of phytol is not dependent on genetic background. Arthritis-preventing effect of phytol when injected (200 μl SC) 5 d before induction of PIA in LEW.1F rats (<i>n</i> = 9).</p> <p>(F) Phytol has an arthritis-ameliorating effect in rats that have a functional oxidative burst. Rats heterozygous for the functional <i>Ncf1</i> (<i>Ncf1</i>^E3) allele with an arthritis susceptibility locus on Chromosome 6 were injected IP (200 μl) with phytol at the estimated peak of disease (day 24, arrow; control, <i>n</i> = 13; phytol, <i>n</i> = 15).</p> <p>(G) Histamine dihydrochloride reverses the ameliorating effect of phytol, possibly by blocking oxidative burst. Injection of histamine twice (200 μl SC and IP), with 5 d between injections (arrows), starting 10 d after phytol treatment (day 26 after pristane) in DA.<i>Ncf1</i>^DA rats could reverse the arthritis-ameliorating effect (<i>n</i> = 5). Results are presented as difference in score compared to day of treatment (day 0).</p> <p>*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p></div

T cells are not affected differently in NCF1*^/* compared to NCF1*^/+ tumor bearing mice with regard to their CD4/CD8 ratio, activation status or memory phenotype.

<p>(A) T cells were gated as singlet cells that were not stained for dead cell marker but positive for CD3. (B) The ratio of CD4+ to CD8+ was gated in blood, spleen and tumor in NCF1*^/* and NCF1*^/+ mice, with the dotted line representing the ratio found in WT C57Bl/6 spleenocytes. (C) Activation marker CD69 was stained on blood, spleen and tumor resident T cells. (D) Blood, spleen and tumor resident T cells were stained for CD62L as well as CD44 to indicate their memory phenotype.</p