Antonio Citro,1,2 Andrea Valle,1 Elisa Cantarelli,1 Alessia Mercalli,1 Silvia Pellegrini,1

Daniela Liberati,1 Luisa Daffonchio,3 Olga Kastsiuchenka,3 Pier Adelchi Ruffini,3

Manuela Battaglia,1 Marcello Allegretti,3 and Lorenzo Piemonti1

CXCR1/2 Inhibition Blocks and ReversesType 1 Diabetes in MiceDiabetes 2015;64:1329–1340 | DOI: 10.2337/db14-0443

Chemokines and their receptors have been associatedwith or implicated in the pathogenesis of type 1diabetes (T1D), but the identification of a single specificchemokine/receptor pathway that may constitutea suitable target for the development of therapeuticinterventions is still lacking. Here, we used multiplelow-dose (MLD) streptozotocin (STZ) injections andthe NOD mouse model to investigate the potency ofCXCR1/2 inhibition to prevent inflammation- andautoimmunity-mediated damage of pancreatic islets.Reparixin and ladarixin, noncompetitive allostericinhibitors, were used to pharmacologically blockadeCXCR1/2. Transient blockade of said receptors waseffective in preventing inflammation-mediated damagein MLD-STZ and in preventing and reversing diabetes inNOD mice. Blockade of CXCR1/2 was associated withinhibition of insulitis and modification of leukocytesdistribution in blood, spleen, bone marrow, and lymphnodes. Among leukocytes, CXCR2+ myeloid cells werethe most decreased subpopulations. Together theseresults identify CXCR1/2 chemokine receptors as“master regulators” of diabetes pathogenesis. Thedemonstration that this strategy may be successful inpreserving residual b-cells holds the potential to makea significant change in the approach to management ofhuman T1D.

Type 1 diabetes (T1D) originates from an immuno-logic disorder that leads the immune system to attackpancreatic b-cells (1). At the onset of T1D, the inflamma-tory response is thought to be directed toward pancreaticislets (2), a process that seems to play a crucial role inmaintaining the autoimmune response as well as repre-senting a key feature of its pathology (3). Consequently,control of the inflammatory response is a strategic and

somewhat underexplored action for influencing the dis-ease (4–7).

Chemokines are a family of small chemoattractantcytokines that control through their receptors a widevariety of physiological and pathological processes, rang-ing from immune surveillance to inflammation, from viralinfection to cancer (8–10). A logical model for initiating/maintaining leukocyte infiltration into mouse and humanpancreatic islets is the secretion, by b-cells themselves, ofchemokines favoring leukocyte recruitment (11–14). Forinstance, b-cell–derived CCL2 recruits monocytes andmacrophages into pancreatic islets (15–18) and CXCL10secreted by insulin-producing cells promotes T-cell infil-tration (19,20).

We recently demonstrated that the inhibition ofCXCR1/2 chemokine receptors is crucial for improvingboth human and murine islet survival after transplanta-tion (21). In this study we hypothesized that CXCR1/2inhibition may also be functional/effective in preventinginflammatory damage to pancreatic islets during diabetesdevelopment. In fact, pancreatic islets produce and se-crete the CXCR1/2 chemokine ligands (named CXCL8,CXCL1, and CXCL2) in response to proinflammatorycytokines (12,15,17,22,23). Furthermore, the concen-tration of CXCR1/2 ligands is elevated in the blood ofboth rodents and humans with autoimmune diabetes(24–26); most important, recent reports support thenotion that neutrophils (the major target of CXCR1/2inhibitors) play a key role in the etiopathogenesis ofT1D (27–29). We therefore extensively characterized theconsequences of CXCR1/2 inhibition on inflammation- andautoimmunity-mediated diabetes in preclinical models.Reparixin (30) and ladarixin (31), CXCR1/2 noncompet-itive allosteric inhibitors that have completed phase I

1San Raffaele Diabetes Research Institute, IRCCS San Raffaele Scientific Institute,Milan, Italy2Department of Surgery, University of Pavia, Pavia, Italy3Research and Development Department, Dompè Farmaceutici S.p.A, L’Aquila, Italy

Corresponding author: Lorenzo Piemonti, piemonti.lorenzo@hsr.it.

Received 18 March 2014 and accepted 6 October 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0443/-/DC1.

© 2015 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

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IMMUNOLOGYAND

TRANSPLANTATIO

N

studies and already entered phase II/III trials (32), wereused to obtain the pharmacologic blockade of the CXCL1–CXCR1/2 axis. The demonstration that this strategy maybe successful in preserving residual b-cells has the poten-tial to make a significant change in the approach to man-aging human T1D.

RESEARCH DESIGN AND METHODS

MiceMale C57BL/6 and female NOD/Ltj mice were purchasedfrom Charles River Laboratories (Calco, Italy). All micewere bred and housed in specific pathogen-free con-ditions. The animals had free access to tap water andpelleted food throughout the course of the study. Allexperiments were in accordance with protocols approvedby the Animal Care and Use Committee of San RaffaeleScientific Institute.

Drugs and TreatmentsReparixin (30) and DF1726A (structurally related toreparixin but not active on CXCR1/2 receptors at 1025–1028 mol/L) were provided by Dompè Farmaceutici S.p.A(L’Aquila, Italy) and administered by continuous subcutane-ous infusion (Alzet osmotic pump; Alza Corporation, PaloAlto, CA) at a dose of 5.4 mg/h/kg for 7 days starting atday 21; ladarixin (31) was provided by Dompè FarmaceuticiS.p.A and administered orally at a dose of 15 mg/kg dailyfor 14 days starting at day 21 or +5; 0.9% sodiumchloride solution was used as vehicle.

Multiple Low-Dose Streptozotocin Injected MouseModelMale C57BL/6 mice received treatment with multiple lowdoses (MLDs) of streptozotocin (STZ). STZ (Sigma-Aldrich,St. Louis, MO) was injected intraperitoneally at a dose of40 mg/kg/day. This was carried out for 5 consecutive days.Nonfasting glucose concentrations in venous blood weremeasured every day starting from the first day of treat-ment. Blood glucose measurements were performed usingan Ascensia Elite XL blood glucose meter (Bayer, Toronto,Ontario, Canada). Mice with nonfasting glycemia $250mg/dL on two consecutive tests were considered diabetic;the first detection of hyperglycemia was considered thedate of diabetes onset. Mice were followed for up to 60days after the first STZ injection.

NOD Mouse ModelFor studies of T1D prevention, female NOD/Ltj micereceived ladarixin or vehicle by oral administration at 4, 8,or 12 weeks of age. Mice were monitored for bloodglucose values twice a week. Diabetes was defined as twoconsecutive nonfasting blood glucose concentrations$250 mg/dL separated by 24 h. For studies of T1D di-abetes reversion, female NOD mice with recent-onset di-abetes received ladarixin or vehicle by oral administration.Animals were monitored two to three times per week forup to 10 weeks, with no exogenous insulin treatment. Re-mission of the disease in treated mice was defined as twoconsecutive glucose measurements ,200 mg/dL. Relapse

of the disease in cured mice was considered following twoconsecutive glucose measurements of $250 mg/dL.

HistologyFemale NOD mice were killed at the end of the treat-ment. Collected pancreata were fixed in 10% bufferedformalin and processed routinely for histology. Histologyslides were stained with hematoxylin and eosin, anti-CD3(rat antihuman CD3 IgG1 with mouse cross-reactivity,clone CD3–12, 1:1000; Serotec, U.K.), anti-F4/80 (ratanti-mouse F4/80 IgG2b, clone A3–1, 1:200; Serotec),and anti-myeloperoxidase (MPO; polyclonal rabbit antihu-man; Dako, Denmark). Antigen was retrieved with TrisEDTA (pH 9) for anti-CD3 and MPO immunostainingand with pronase Bond Enzime diluent kit (Leica, Italy)for anti-F4/80 immunostaining. The degree of islet infil-tration was analyzed by two independent observers whowere blinded to the experimental conditions. Each ob-server assessed a minimum of 40 islets per animal. Insu-litis was scored as previously described (33): 0, noinsulitis; 1, peri-insulitis; 2, mild insulitis (,25% of infil-trated islets); 3, severe insulitis (25–75% of infiltratedislets). In terms of infiltration, for each parameter wealso evaluated the morphology of the islet: 0, physiolog-ical islet; 1, amorphous islet. The total score is the addi-tion of the infiltration and the morphological scores. Theleukocyte infiltration score (CD3+ and F4/80+ cells) wasquantified as follows: 0, no infiltration; 1, low peri-isletinfiltration; 2, mild peri-islet infiltration; 3, high intraisletinfiltration (Supplementary Fig. 1). Neutrophils infiltra-tion was evaluated as the number of MPO+ cells per islet.

Flow CytometrySingle-cell suspensions were prepared from differenttissues (i.e., pancreas, pancreatic lymph nodes, spleen,bone marrow, and blood) and were stained at 4°C in PBScontaining 2% FBS and 0.5% EDTA. Cell surfaces werestained with fluorescein isothiocyanate–, phycoerythrin-,or allophycocyanin-labeled anti-CD4, anti-CD8 (clone 53–6.7), anti-CD3 (clone 145–2C11), anti-CD19 (clone 1D3),anti-Gr-1 (clone RB6–8C5), anti-CD11b (clone M1/70),anti-Ly6G (clone 1A8), anti-Ly6C (clone AL-21; BD Bio-sciences, San Diego, CA), and anti-CXCR2 antibodies(clone 242216; R&D Systems, Minneapolis, MN). BothGr-1+Ly6C2CD11b+ and Ly6G+CD11b+ combinations wereused to identify polymorphonuclear cells (PMNs). Linearregression analysis showed no statistically significant dif-ference between the two strategies used for neutrophilidentification (95% CI of the slope 0.9874–1.121) (datanot shown). Consequently, on the basis of the expression ofthe mentioned markers, Gr-1+CD11b+Ly6C2 (mostly PMNs),CD3+CD4+ (mostly T-helper cells), CD3+CD8+ (mostly cyto-toxic T lymphocytes), CD3+CD42CD82 (mostly NKT cells),CD19+ (mostly B lymphocytes), Gr-12CD11b+Ly6C2 (mostlynatural killer cells), and Gr-12CD11b+Ly6C+ (mostly mono-cytes/macrophages) cells were identified. To exclude a pos-sible contamination of T cells within the gate of MF, Gr-1and Ly6C markers were evaluated on CD3+ and CD19+ cells.

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The frequencies of CD3+ and CD19+ cells that decreasewithin the macrophage gate were irrelevant (0.08% and0.26%, respectively; data not shown). Samples were acquiredon a FACSCantoII instrument (BD Biosciences). Rainbowcalibration particles (Spherotech Inc., Lake Forest, IL)were used to calibrate and normalize acquisition settingsin each experiment. Flow cytometry data were analyzedwith FCS Express V4 (DeNovo Software, Glendale, CA).

Intrapancreatic Leukocyte IsolationCollagenase IV (Sigma-Aldrich, St. Louis, MO) solution(2 mg/mL; 2.5 mL) was injected into the bile duct. Pancreassamples were collected and digested at 37°C for 20 min.Obtained digested tissue was washed with cold RPMI 1640medium and FBS (Lonza, Belgium) and resuspended ina mixture of Percoll (GE Healthcare/Biosciences AB, Swe-den) and Histopaque 1077 (Sigma-Aldrich). After gradientcentrifugation (2,000 rpm for 20 min), intrapancreaticleukocytes were collected from the interface. The collectedcells were washed twice with RPMI 1640 medium andused for further analysis.

Cytokine Multiplex AnalysisSerum samples were analyzed for 23 cytokines using theBio-Plex Pro Mouse Cytokine Standard 23-plex (group 1;Bio-Rad Laboratories, Hercules, CA) according to themanufacturer’s protocol.

Statistical AnalysisVariables are summarized as mean 6 SD or median andinterquartile range according to their distribution. Vari-ables with a normal distribution were compared with one-way unpaired Student t test (two groups). Variables with anon-normal distribution were compared using the Mann-Whitney U test. Categorical variables were comparedusing the x2 test or Fisher exact test, as appropriate.The cumulative diabetes incidence or reversion was eval-uated using Kaplan-Meier analysis, and the significancewas estimated using the log-rank test. Statistical analy-sis of nonfasting glycemia during follow-up was per-formed using the general linear model for repeatedmeasures. Hazard ratios for new-onset diabetes or re-versed diabetes after treatment were estimated usingCox regression. All statistical analyses were performedusing SPSS statistical software version 13.0 (SPSS Inc.,Chicago, IL).

RESULTS

Pharmacologic Blockade of CXCR1/2 PreventsDiabetes Induction in a Model of Inflammation-Mediated Islet DestructionTo determine the efficacy of CXCR1/2 inhibition as astrategy to prevent inflammation-mediated islet destruction,we tested the ability of two different CXCR1/2 inhibitors toprevent diabetes induction after MLD STZ injections. In thefirst set of experiments we tested reparixin, a noncompeti-tive allosteric CXCR1/2 inhibitor (30) that must be admin-istered via continuous subcutaneous infusion because of itsshort half-life. A total of 34 male C57BL/6 mice were

intraperitoneally injected with MLD STZ (40 mg/kg/dayfor 5 days), and reparixin (8 mg/h/kg; n = 17) or vehicle(n = 17) was administered starting from day21 up to day16 after the first STZ injection. Reparixin treatment signifi-cantly prolonged the timing of diabetes development. Themedian diabetes-free time was 12 6 0.6 and 7 6 0.6 daysfor reparixin- and vehicle-treated mice, respectively (P =0.001; Fig. 1A). More important, glycemia remained con-stantly and significantly lower in the reparixin-treated groupthan in the vehicle-treated group after diabetes developed(P = 0.039; Fig. 1B). Notably, DF1726A, a compound struc-turally related to reparixin but not active on CXCR1/2(34), did not prevent diabetes development when admin-istered in the same experimental conditions (Supplemen-tary Fig. 2A). Moreover, reparixin treatment at 15 mg/kgthree times a day per os (a dose able to inhibit selectivelyCXCR1 but not CXCR2) did not prevent diabetes (Supple-mentary Fig. 2B).

In the second set of experiments we tested ladarixin,a potent blocker of both CXCR1 and CXCR2 and suitablefor chronic oral administration because of its longer invivo half-life (35). In a total of 52 male C57BL/6 mice thatreceived MLD STZ treatment (Fig. 1C), ladarixin treat-ment significantly prolonged the duration of diabetes de-velopment, and its efficiency was strictly dependent onthe time when treatment was started and the duration oftreatment (Fig. 1C and D). The median diabetes-free timewas 10 6 2.5 days for vehicle-treated mice (n = 12). Whenstarted on day 21, the median diabetes-free time was13 6 2 days (P = 0.22 vs. control, n = 8) and 29 6 16days (P = 0.002 vs. control, n = 8), respectively, for 7 (datanot shown) and 14 days of ladarixin treatment. Whentreatment with ladarixin for 14 days was started atday +5 or +10 after the first STZ injection, the mediandiabetes-free time was 16 6 3 days (P = 0.031 vs. control,n = 12) and 10 6 1 days (P = 0.61 vs. control, n = 12),respectively (data not shown). Similar to reparixin treat-ment, glycemic levels remained constantly and signifi-cantly lower in the ladarixin-treated group than in thevehicle-treated group after diabetes developed (Fig. 1D).

Pharmacologic Blockade of CXCR1/2 PreventsInsulitis and Diabetes in the NOD MouseTo determine whether pharmacologic blockade of CXCR1/2prevents the development of spontaneous diabetes in NODmice, female mice were randomized to receive ladarixin orvehicle for 2 consecutive weeks starting in the early or latepreclinical stage of the disease.

Ladarixin significantly delayed and prevented diabetesonset when administered at 12 weeks of age (P = 0.007;Fig. 2A), but not at 4 weeks of age, even if a trend wasevident (P = 0.175). Specifically, 22% of the mice receivingCXCR1/2 inhibitor from 12 weeks of age developed di-abetes during the follow-up (i.e., 62 weeks of age; meantime to diabetes 25.8 6 9.4 weeks), whereas 78% of thevehicle-treated mice developed diabetes during the sameperiod (mean time to diabetes 17.8 6 4.1 weeks; Fig. 2A).

diabetes.diabetesjournals.org Citro and Associates 1331

As for 12-week-old NOD mice, treatment of 8-week-oldmice resulted in significantly delayed diabetes onset (P =0.046; data not shown).

To determine the progression of islet mass loss andinsulitis in NOD mice and the effect of CXCR1/2 blockadeon such events, we analyzed islet numbers and insulitis inladarixin- and vehicle-treated mice at the end of thetreatment (Fig. 2B). There was no significant difference inislet numbers between ladarixin- and vehicle-treatedgroups or between 4- and 12-week-old mice. However,insulitis score (Fig. 2C) and infiltrating CD3+ T cells,F4/80+ macrophages, and MPO+ neutrophils were signif-icantly skewed toward lower values (Fig. 3).

Pancreas-infiltrating leukocytes were isolated from12-week-old mice and characterized by flow cytometry(CD45, Gr-1, Ly6C, CD11b, CXCR2) at the end of thetreatment. The number of leukocytes was significantlysmaller in ladarixin-treated than in vehicle-treated mice(250,000 6 57,735 vs. 440,000 6 89,814 cells/pancreas;P = 0.012). Among all CD45+ cells, CXCR2+ cells were lessabundant in ladarixin-treated than in vehicle-treated mice(17.8 6 5.1% vs. 10.7 6 2.3%; P = 0.045) (SupplementaryFig. 3). CD45+ and CD45+CXCR2+ cells were analyzed sepa-rately for their expression of Gr-1, Ly6C, and CD11b (Fig. 4A

and B). Among CD45+, lymphoid cells (Gr-12Ly6C2CD11b2)were the most abundant leukocytes (656 17%), followed bymacrophages (Gr-12Ly6C+CD11b+, 23 6 7%) and neutro-phils (PMNs; Gr-1+Ly6C2CD11b+, 1.5 6 1.1%). Ladarixintreatment significantly reduced the percentage of PMNsand the absolute number of PMNs and lymphoid cells.

Among CD45+CXCR2+cells, macrophages (94 6 3%)were the most abundant, followed by lymphoid cells(4 6 2%) and PMNs (1.3 6 1.3%). Ladarixin treatmentsignificantly reduced the absolute number of macrophages.

Pharmacologic Blockade of CXCR1/2 in NOD Mice:ImmunophenotypingCD45+ leukocytes were analyzed by flow cytometry indifferent tissues (i.e., blood, spleen, pancreatic lymphnodes, and bone marrow) after 14 days of vehicle orladarixin treatment. The analysis showed myeloid cellsand, in particular, PMNs, readily identified by expressionof Gr-1 and/or Ly6C, as the predominant CXCR2+ cellsamong blood, spleen, and bone marrow leukocytes (Sup-plementary Table 1). CXCR2 is also expressed, dependingon the tissue context, by part of CD42CD82 double neg-ative CD3+ lymphocytes and by few CD19+ B lymphocytesand CD3+CD4+ or CD3+CD8+ T lymphocytes.

Figure 1—CXCR1/2 blockade by reparixin or ladarixin modulates the induction of diabetes after MLD STZ injections. Male C57BL/6 micereceived MLD STZ treatment. STZ was injected intraperitoneally at a dose of 40 mg/kg/day for 5 consecutive days. Reparixin (dose of 5.4mg/h/kg; n = 17) or vehicle (n = 17) treatment was administered by continuous subcutaneous infusion starting from day 21 up to day +6after the first STZ injection. Alternatively, ladarixin (15 mg/kg/day) or vehicle treatment (n = 12) was administered orally starting from day21up to day +13 (n = 8) or from day +5 up to day +19 (n = 12) after the first STZ injection. A and C: Kaplan-Meier analysis of diabetes-freesurvival. Differences were tested using the log rank statistic. B and D: Nonfasting glycemia during the 60-day follow-up. Gray areasrepresent the treatment windows. Data are expressed as box plots. Statistical analysis was performed by general linear model for repeatedmeasures.

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In 4-week-old mice (Supplementary Table 2) theCXCR1/2 inhibitor induced mild modification of whiteblood cells, whereas spleen and pancreatic lymph nodeswere unaffected. A significant increase in total circulatingleukocytes was evident and, among CD45+ cells, the pro-portions of CXCR2+ cells and PMNs increased.

In 12-week-old mice (Supplementary Table 2), majorand more extensive changes were observed. Total bloodcirculating leukocytes were greatly increased; cellularitywas augmented in pancreatic lymph nodes and reducedin bone marrow. Ladarixin treatment decreased signifi-cantly the proportion of CD45+CXCR2+ cells in bloodand lymph nodes. At least one of the myeloid cell frac-tions (Gr-1+Ly6C2CD11b+, Gr-12CD11b+Ly6C+) was con-sistently reduced by CXCR1/2 inhibition in blood, spleen,and bone marrow. A surprising increase in CD45+/CD19+

B lymphocytes was evident in blood and spleen, whereasladarixin treatment differed in its effects on T lymphocytesdepending on site: fractional expression of CD45+/CD3+

T lymphocytes increased in blood and bone marrow anddecreased in spleen. CD4+CD25+Foxp3+ regulatory T cells in

blood and spleen were not affected by CXCR1/2 treatment(data not shown).

We also compared the levels of systemic cytokinesbetween vehicle- and ladarixin-treated mice at differentages. With the exception of a mild significant decrease ofinterleukin (IL)-9 and IL-6 in 4- and 12-week-old micetreated with the CXCR1/2 inhibitor, respectively, thecytokine profiles did not change (Supplementary Table 3).

CXCR1/2 Inhibitor Reverses Diabetes in Diabetic NODMiceNOD female mice with recent-onset diabetes receivedeither ladarixin (15 mg/kg/day; n = 28) or vehicle (n = 22)for 14 days. Strikingly, diabetes was reversed in 22 of 28ladarixin-treated NOD mice (78%) (Fig. 5A). Remissionwas rapid (detected in 20 of 22 NOD mice [91%] within72 h of treatment), and 5 of 22 remission NOD mice(23%) remained diabetes free .10 weeks after onset(Fig. 5B). The majority of NOD mice receiving vehicle,however, failed to undergo remission or, if induced (2of 22 [9%]; P , 0.001), remission was short (7 and 17

Figure 2—Blockade of CXCR1/2 inhibits insulitis and the development of T1D in NOD mice. Female NOD mice were treated with ladarixin(15 mg/kg/day per os) or vehicle for 14 days at 4 (4w) or 12 weeks (12w) of age, and incidence of diabetes was monitored. A: Kaplan-Meieranalysis of diabetes-free survival. Blood glucose concentrations were monitored twice a week. Diabetes incidence was defined based ontwo consecutive glucose measures $250 mg/dL. Differences were tested using the log rank statistic (n = 14 mice/group). Gray areasrepresent the treatment windows. B: Number of islets per area after 2 weeks of vehicle (n = 4) or ladarixin treatment (n = 4) at variousages. Data are expressed as box plots (number of islets per area) and analyzed using the Mann-Whitney U test. C: Insulitis score after 2 weeksof vehicle (n = 4) or ladarixin treatment (n = 4) (left panel) and representative pancreas stained with hematoxylin and eosin (original magni-fication 320) (right panel). Data are expressed as a histogram and analyzed using the x2 test.

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days). Diabetes reversion was not associated with mod-ification of CD4+CD25+Foxp3+ regulatory T cells in bloodand spleen (data not shown) or systemic cytokine profilechanges, with the exception of a mild IL-12p70 increase(data not shown).

Notably, the average blood glucose concentration inladarixin-treated mice remained significantly lower thanthat of vehicle-treated mice during the entire follow-up(Fig. 5C). Lower glucose concentrations at diabetes onsetcorrelated with a higher efficacy of ladarixin treatment.In fact the CXCR1/2 inhibitor maintained the ability toreverse diabetes (43%) in highly hyperglycemic NODmice (glucose concentrations $350 mg/dL), but diabetes

reversion did not demonstrate a long-term benefit (Sup-plementary Fig. 4). Similarly, late diabetes onset (.21weeks of age) did not affect ladarixin-induced reversionof diabetes but correlated with an earlier recurrence ofthe disease (Supplementary Fig. 4).

Histological analysis was performed to determine theprogression of islet mass loss and insulitis and the effectof CXCR1/2 blockade on such events (Fig. 6). According toblood glucose concentration, a significantly higher num-ber of islets were counted in the pancreas of ladarixin-treated mice compared with vehicle-treated mice and,among ladarixin-treated mice, in the pancreas of micewith diabetes remission. Both insulitis (Fig. 6) and

Figure 3—Pancreas-infiltrating neutrophils, macrophages, and T cells in vehicle- and ladarixin-treated NOD mice. Female NOD mice weretreated with ladarixin (15 mg/kg/day per os) or vehicle for 14 days at 4 (4w; n = 4) or 12 weeks (12w; n = 4) of age. Left panels:Representative pancreas immunohistochemistry staining for MPO, F4/80, and CD3 after 2 weeks of vehicle or ladarixin treatment (originalmagnification 320). Right panels: Neutrophils were quantified as MPO-positive cells per islet, and grade of F4/80+ and CD3+ cellsinfiltration were expressed as leukocyte infiltration score. Data are expressed as histograms (leukocyte infiltration score) or box plot (cellsper islet) and analyzed using the Mann-Whitney U and x2 tests, respectively.

1334 CXCR1/2 and Type 1 Diabetes Diabetes Volume 64, April 2015

leukocyte infiltration scores (Fig. 7) were significantly de-creased toward lower values in ladarixin-treated mice.

DISCUSSION

We recently reported that the inhibition of CXCR1/2chemokine receptors is relevant for improving humanand murine islet survival after transplantation (21).Here we demonstrate that the same pathway seemsto be relevant for islet survival after inflammation-and autoimmunity-mediated damage in two preclinicalmodels. To our knowledge this is the first study demon-strating success in preventing and reversing diabetes byinhibiting CXCR1/2 chemokine receptors, identifyingthis pathway as a “master regulator” of diabetes patho-genesis. Concordantly, Diana and Lehuen (36) recentlyreported that treatment with the CXCR2 antagonistSB225002 at early ages dampens the later developmentof autoimmune diabetes in NOD mice. Even if SB225002seemed to be ineffective when used at a late age (36), theresults strongly support the therapeutic potential ofCXCR1/2 chemokine receptors. More than one-half ofthe ;50 human/mouse chemokines have been previ-ously associated with, or implicated in, the pathogenesis

of T1D (12), but the identification of a single specificchemokine/receptor pathway that may constitute a suit-able target for the development of therapeutic interven-tions is still lacking (37). Chemokines such as CCL2,CCL3, CCL5, CCL22, and CXCL10 and their receptorswere suggested to directly affect T1D development.However, disease prevention in the NOD model can beachieved only by interfering with more than one of them(37,38). Individual treatments reduced inflammatoryinfiltrates without effectively preventing diabetes devel-opment in some experimental settings (12) or evenworsening the disease in others. For example, NODmice deficient in CXCR3 (CXCL19/CXCL10 receptor) orCCR5 (CCL3/CCL4/CCL5 receptors) developed sponta-neous diabetes earlier than wild-type NOD mice(39,40).

In our study we established a relationship involvingthe CXCR1/2 chemokine receptors and the developmentof insulitis and diabetes in mice. We first demonstratedthat transient CXCR1/2 inhibition prevents inflammation-mediated islet damage in the MLD STZ model. MLDSTZ–induced b-cell destruction is associated with a cell-dependent inflammatory reaction and with a central

Figure 4—Flow cytometry of intrapancreatic leukocytes. Female NOD mice were treated with ladarixin (15 mg/kg/day per os; n = 4)or vehicle (n = 4) for 14 days at 12 weeks and intrapancreatic leukocytes were obtained as described in RESEARCH DESIGN AND METHODS.A: Representative flow cytometry dot plot and histogram after CD45, CXCR2, Gr-1, Ly6C, and CD11b staining. Within live leukocytes(CD45+), lymphoid cells (Ly), macrophages (MF), and neutrophils (PMN) were identified as Gr-1-Ly6C-, Gr-1-Ly6C+CD11b+, and Gr-1+Ly6C-CD11b+ cells, respectively. B: Frequency and absolute number of PMN, Ly, and MF. Data are expressed as box plots. *P <0.05, Mann-Whitney U test.

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pathogenic role of proinflammatory cytokines, such asIL-1b and interferon-g (41), but does not require func-tional T and B cells (42). In this model two differentCXCR1/2 inhibitors were effective in protecting islets,whereas a nonactive, structurally related compound wasnot, confirming that the effect was not caused by multipleoff-target actions. Second, we demonstrated that tran-sient CXCR1/2 blockade inhibits autoimmune insulitisin NOD mice, clearly reducing neutrophil, macrophage,and T-cell infiltration. This effect occurred independentof age and, starting from 8 weeks, was associated with theprevention of autoimmunity-mediated islet damage. Ofnote, even if batches of control and treatment groupsrun in parallel at all ages, the rate of T1D onset in4-week-old NOD mice in the control group was relativelylower than in the 12-week-old mice, and this pattern mayhave contributed to reducing the impact of the interven-tion started at the earliest age. Third, we demonstratedthat transient CXCR1/2 inhibition is able to reverse di-abetes, preserving islet mass after onset (21). This is inagreement with indirect evidence previously reported.Human pancreatic islets produce and secrete the proin-flammatory CXCL8 and CXCL1 ligands (12,15,17,22,23),whereas lipopolysaccharide-induced production of CXCL8by neutrophils is increased in prediabetic and T1Dpatients. In parallel, circulating concentrations of CXCL8are elevated in children with T1D compared with

nondiabetic controls (24–26). Specifically, concentrationsof CXCL8 correlate with glycemic control; higher concen-trations are associated with poor or inadequate glucosecontrol. Finally, and most important, recent reports dem-onstrate that neutrophils, the major target of CXCR1/2inhibitors, play a key role in the etiopathogenesis of T1Din mice and humans (27–29,36).

The CXCR1/2 noncompetitive allosteric inhibitors usedin our work have different pharmacologic characteristics(31). Ladarixin is a potent blocker of both CXCR1 andCXCR2, with a half-maximal inhibitory concentration inthe range of 1 to 2 nmol/L (31). In contrast, reparixinexhibits ;400-fold greater efficacy in blocking CXCR1compared with CXCR2 (30). We imputed the effect mainlyto the inhibition of CXCR2 and not of CXCR1 in mice.Reparixin treatment at a dose able to inhibit mainlyCXCR1, but not CXCR2, was unable to prevent diabetesin the MLD STZ model. Moreover, despite recent studiesreporting the existence of a mouse homolog of humanCXCR1 (43–45), none of the known CXCR1/2-related che-mokines have been shown to activate this putative recep-tor (43–45). Consequently, mice are considered to haveonly functional CXCR2 and, although we cannot com-pletely exclude a possible action mediated by the inhibi-tion of some not yet discovered CXCR1 functions, CXCR2should be considered as the unique target of CXCR1/2inhibitors in mice.

Figure 5—Short-course treatment with the CXCR1/2 inhibitor rapidly induces remission in recent-onset diabetic NOD mice, which ismaintained during follow-up. NOD female mice with recent-onset diabetes (two consecutive nonfasting blood glucose concentrations$250 mg/dL) were treated with ladarixin (15 mg/kg/day per os; n = 28) or vehicle (n = 22) for 14 days and blood glucose was monitored.Kaplan-Meier and Cox regression analyses are shown of diabetes remission (A) and recurrence (B). Blood glucose concentrations weremonitored twice a week. Differences were tested using the log rank statistic and hazard ratios are shown. C: Nonfasting blood glucoseconcentrations of recent-onset diabetic NOD mice before and after treatment. Data are expressed as box plots. *P < 0.05, **P < 0.01,Mann-Whitney U test.

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To further investigate the protective mechanism(s)mediated by the CXCR1/2 inhibitor in NOD mice, weanalyzed CXCR2 expression, systemic leukocytes by flowcytometry, and the systemic cytokine profile by a mul-tiplex approach. CXCR2 was confirmed to be highlyexpressed on myeloid cells. Nevertheless, CXCR2 alsowas detected on lymphoid cells (natural killer T cells,B cells, and CD8+ and CD4+ T cells). In concordance withthis, intrapancreatic myeloid CD45+CXCR2+ cells weresignificantly reduced by CXCR1/2 blockade. Other signif-icant modifications observed in the later phase of thedisease involved not only the myeloid subpopulation inbone marrow and spleen but also the circulating lymphoidsubpopulation. Of note is the observation of increasedB cells in ladarixin-treated NOD mice. The role of B cellsin the modulation of islet immune cell responses has beenrecently recognized in models of autoimmune diabetes(46,47) and immunomodulatory effects of Gr-1+CD11b+

cells were reported in NOD mice receiving anti-CD20treatment (48). In addition, IL-9, IL-6, and IL-12p70were modified by CXCR1/2 inhibitor treatment in relationto the different disease phases. Currently, determiningwhich of these modifications have the most relevantcausal relationship with the prevention of diabetes is

not possible. Moreover, we cannot exclude that diabetesprevention could be caused by the combined activities ofseveral leukocyte subpopulations. The picture is furthercomplicated by the fact that the CXCR1/2 ligands are notlimited to CXCL1/2/8 produced by islets but also includeCXCL3/5/7 in mice (which lack CXCL8) and CXCL3/5/6/7,as well as some other ligands, in humans (49). Additionalstudies, including selective CXCR2 knockout in leukocytepopulations, are required to ascertain the impact of in-dividual changes. Since depletion of neutrophils, the ma-jor CXCR2-positive circulating leukocyte population, hasrecently been suggested to reduce spontaneous T1D in-cidence in NOD mice (36), we can speculate that theycould constitute the more relevant target of CXCR1/2inhibitors. On this basis preliminary experiments withanti-Ly6G, a neutrophil-depleting agent, in associationor comparison with the CXCR1/2 inhibitor, were per-formed both in the MLD STZ model and 12-week-oldNOD mice (data not shown). Anti-Ly6G alone was ineffi-cient and combination with the CXCR1/2 inhibitor was asefficient as the CXCR1/2 inhibitor alone in preventingdiabetes. These results suggest that the action of theCXCR1/2 inhibitor on neutrophils could be necessarybut not sufficient to prevent diabetes in these models.

The results we obtained in preclinical models offerimportant insights in the context of the literature andsuggest possible avenues and expectations in future clinicaltrials. Preserving residual b-cell function has been associ-ated with a reduced rate of microvascular complicationsand hypoglycemia, an improved quality of life, and anoverall reduction in morbidity and disease-associatedmanagement costs (50). Therefore, approaches aimed atcontrolling the autoimmune response and restoring self-tolerance to pancreatic b-cells have attracted much clinicaland scientific interest (6). Among the many pharmacologicinterventions directed at this purpose, rituximab (51),abatacept, CD3-specific monoclonal antibodies (tepluzi-mab, otelixizumab) (52), GAD65 (Diamyd) (53), and HSPp277 (DiaPep) have progressed to phase IIB/III clinicaltrials (7). Other agents, including cytokine modulators(e.g., anti-tumor necrosis factor– or anti-IL-1–basedagents) (54) are also under consideration for large-scaleevaluation. Unfortunately, despite some positive outcomes,none of the pharmacologic approaches tested thus farhave resulted in a consensus agreement that acknowl-edges an acceptable means for preserving C-peptideafter diabetes onset. In addition, when these trials areconsidered together, it seems that therapies based ona single drug are unlikely to have substantial effects onthe prevention or amelioration of hyperglycemia. Ourdata support the idea that the inclusion of CXCR1/2inhibitors in future trials of multiple-drug regimens forT1D may provide therapeutic benefit by preserving b-cellmass. Interestingly, in the current clinical experience,ladarixin was already tested in humans in three phase Ipharmacokinetic/safety studies, including repeated oraladministrations, and it was safe and well tolerated. On

Figure 6—Blockade of CXCR1/2 inhibits insulitis after the develop-ment of T1D in NOD mice. Recent-onset diabetic NOD female mice(two consecutive nonfasting blood glucose concentrations >250mg/dL) were treated with ladarixin (15 mg/kg/day per os) or vehicle.After 14 days, 4 vehicle-treated mice, 4 ladarixin-treated mice with-out diabetes (reverted), and 4 ladarixin-treated mice with diabetes(not reverted) were killed for pancreas analysis. A: Insulitis scoreand islet number per area after 2 weeks of vehicle (n = 4) or ladarixintreatment (n = 8). Data are expressed as histograms (insulitis) or boxplots (islet number per area) and analyzed using the Mann-WhitneyU and x2 tests, respectively. B: Representative pancreas hematoxylinand eosin staining after 2 weeks of vehicle or ladarixin treatment(original magnification 320).

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this basis a trial of ladarixin in patients recently diag-nosed with T1D will be started in the next months.

In conclusion, we identified CXCR1/2 chemokinereceptors and their ligands as “master regulators” anddemonstrated that CXCR2 blockade could be a usefulstrategy in preventing and/or reversing diabetes in mice.The demonstration that the inhibition of this pathway maybe successful in preserving residual b-cells in patients withnew-onset T1D holds the potential to significantly changeour approach to the management of this disease. Thistreatment, focused on inhibiting inflammatory responseby targeting leukocyte migration, could represent an idealcomplement to any intervention acting on the interdictionof the pathogenic autoimmune process.

Acknowledgments. This work was performed in partial fulfillment of therequirements for a PhD degree for A.V. and E.C.Funding. This research was supported by the European Union (HEALTH-F5-2009-241883-BetaCellTherapy) and was partially funded by Dompè FarmaceuticiS.p.A in the fiscal year preceding the date of submission.Duality of Interest. L.D., O.K., P.A.R., and M.A. are Dompè FarmaceuticiS.p.A employees. L.P. received grants for research by Dompè Farmaceutici S.p.A

in the fiscal year preceding the date of submission and is involved in two clinicaltrials supported by Dompè Farmaceutici S.p.A (NCT01220856 and NCT01817959).No other conflicts of interest relevant to this article were reported.Author Contributions. A.C. and E.C. performed the in vivo mouse stud-ies. A.C. and A.V. performed flow cytometry immunophenotyping. A.C., A.V.,A.M., and S.P. performed the histopathological analysis of the pancreas. D.L.performed multiplex analysis of cytokines. L.D., M.B., and M.A. reviewed andedited the manuscript and contributed to the discussion. O.K. and P.A.R.developed and provided CXCR1/2 inhibitors. L.P. developed the concept,designed the experiments, wrote the manuscript, promoted the study, andresearched data. L.P. is the guarantor of this work and, as such, had fullaccess to all the data in the study and takes responsibility for the integrity ofthe data and the accuracy of the data analysis.

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