Rheumatoid arthritis: Methods for two murine models.

Rheumatoid arthritis is an incurable chronic inflammatory disease for which the pathophysiology is not fully understood, and treatment options are flawed. Thus, animal models are used to dissect disease pathogenesis and to develop improved therapeutics. However, accurately modeling all aspects of human rheumatoid arthritis in mice is not possible, and each model has pros and cons. Two useful murine models of rheumatoid arthritis are collagen induced arthritis and TNF induced arthritis. Both recapitulate the chronic inflammatory, erosive arthritis of human rheumatoid arthritis. Collagen induced arthritis has the added similarity to human rheumatoid arthritis of pathogenic autoantibodies, but can have variable degrees of arthritis severity, a challenge for experiments. In contrast, TNF induced arthritis tends to be uniform, but primarily models the innate arm of the immune response. Here we describe the benefits, limitations, and details for both models to help investigators select and implement an appropriate model to achieve the goals of their experiments.

Peptidylarginine Deiminase 2 in Murine Antiviral and Autoimmune Antibody Responses.

The peptidylarginine deiminases (PADs) and the citrullinated proteins that they generate have key roles in innate immunity and rheumatoid arthritis, an inflammatory arthritis with antibodies that target citrullinated proteins. However, the importance of PADs, particularly PAD2, in the adaptive immune response, both normal and pathogenic, is newly emerging. In this study, we evaluated a requirement for PAD2 in the antibody response in collagen-induced arthritis (CIA), a T and B cell-driven murine model of rheumatoid arthritis, and in the protective antibody response to murine influenza infection. Using PAD2-/- and PAD2+/+ mice on the DBA/1J background, we found that PAD2 is required for maximal anti-collagen antibody levels, but not collagen-specific plasma cell numbers, T cell activation or polarization, or arthritis severity in CIA. Also, we found that PAD2 is required not just for normal levels of persistent hemagglutination inhibiting antibodies but also for full protection from lethal influenza rechallenge. Together, these data provide evidence for a novel modest requirement for PAD2 in a normal antiviral antibody response and in an abnormal autoantibody response in inflammatory arthritis.

Arthritis is inhibited in Borrelia-primed and infected interleukin-17A-deficient mice after administration of anti-gamma-interferon, anti-tumor necrosis factor alpha and anti-interleukin-6 antibodies.

The role that cytokines play in the induction of Lyme arthritis is gradually being delineated. We showed previously that severe arthritis developed in a T-cell-driven murine model, even in mice lacking interleukin-17A (IL-17A) and administered anti-gamma-interferon (IFN-γ) antibody. Increased levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), two pro-inflammatory cytokines, were detected in cultures of popliteal lymph node cells obtained from these mice. We hypothesized that concomitantly administered anti-IL-6, anti-TNF-α and anti-IFN-γ antibodies would inhibit the development of arthritis in IL-17A-deficient mice. Our results showed that swelling of the hind paws and histopathological changes consistent with arthritis were significantly reduced in IL-17A-deficient mice that administered the three anti-cytokine antibodies. These results suggest that treatment with multiple anti-cytokine antibodies can abrogate the induction of Lyme arthritis in mice.

Peptidylarginine deiminase 2 is required for tumor necrosis factor alpha-induced citrullination and arthritis, but not neutrophil extracellular trap formation.

Citrullination, the post-translational conversion of arginines to citrullines, may contribute to rheumatoid arthritis development given the generation of anti-citrullinated protein antibodies (ACPAs). However, it is not known which peptidylarginine deiminase (PAD) catalyzes the citrullination seen in inflammation. PAD4 exacerbates inflammatory arthritis and is critical for neutrophil extracellular traps (NETs). NETs display citrullinated antigens targeted by ACPAs and thus may be a source of citrullinated protein. However, PAD4 is not required for citrullination in inflamed lungs. PAD2 is important for citrullination in healthy tissues and is present in NETs, but its role in citrullination in the inflamed joint, NETosis and inflammatory arthritis is unknown. Here we use mice with TNFα-induced inflammatory arthritis, a model of rheumatoid arthritis, to identify the roles of PAD2 and PAD4 in citrullination, NETosis, and arthritis. In mice with TNFα-induced arthritis, citrullination in the inflamed ankle was increased as determined by western blot. This increase was unchanged in the ankles of mice that lack PAD4. In contrast, citrullination was nearly absent in the ankles of PAD2-deficient mice. Interestingly, PAD2 was not required for NET formation as assessed by immunofluorescence or for killing of Candida albicans as determined by viability assay. Finally, plasma cell numbers as assessed by flow cytometry, IgG levels quantified by ELISA, and inflammatory arthritis as determined by clinical and pathological scoring were all reduced in the absence of PAD2. Thus, PAD2 contributes to TNFα-induced citrullination and arthritis, but is not required for NETosis. In contrast, PAD4, which is critical for NETosis, is dispensable for generalized citrullination supporting the possibility that NETs may not be a major source of citrullinated protein in arthritis.

Borrelia-primed and -infected mice deficient of interleukin-17 develop arthritis after neutralization of gamma-interferon.

The immune mechanisms responsible for development of Lyme arthritis are partially understood with interleukin-17 (IL-17) and gamma-interferon (IFN-γ) playing a generally accepted role. Elevated levels of IL-17 and/or IFN-γ have been reported in samples from human Lyme arthritis patients and experimental mice. In addition, IL-17 and IFN-γ have been implicated in the onset of arthritis in Borrelia-primed and -infected C57BL/6 mice. Recently, we showed that IL-17-deficient mice developed swelling and histopathological changes consistent with arthritis in the presence of high levels of IFN-γ. We hypothesized that neutralization of IFN-γ in IL-17-deficient mice would inhibit Borrelia-induced arthritis. Our results, however, showed that swelling of the hind paws and histopathological changes of arthritis did not differ between Borrelia-primed and -infected IL-17-deficient and wild-type mice with or without neutralization of IFN-γ. We also found higher levels of tumor necrosis factor alpha (TNF-α) and IL-6 in the popliteal lymph node cells of Borrelia-primed and -infected IL-17-deficient mice after neutralization of IFN-γ. These results suggest that multiple cytokines interact in the development of Borrelia-induced arthritis.

Arthritis is developed in Borrelia-primed and -infected mice deficient of interleukin-17.

Interleukin-17 (IL-17) has been shown to participate in the development of Lyme arthritis in experimental mice. For example, neutralization of IL-17 with antibodies inhibits induction of arthritis in Borrelia-primed and -infected C57BL/6 wild-type mice. We hypothesized that mice lacking IL-17 would fail to develop Borrelia-induced arthritis. IL-17-deficient and wild-type C57BL/6 mice were primed with heat-inactivated Borrelia and then infected with viable spirochetes 3 weeks later. No swelling or major histopathological changes of the hind paws were detected in IL-17-deficient or wild-type mice that were primed with Borrelia or infected with viable spirochetes. By contrast, IL-17-deficient and wild-type mice that were primed and subsequently infected with heterologous Borrelia developed severe swelling and histopathological changes of the hind paws. In addition, Borrelia-primed and -infected IL-17-deficient mice exhibited elevated gamma-interferon (IFN-γ) levels in sera and increased frequencies of IFN-γ-expressing lymphocytes in popliteal lymph nodes compared to Borrelia-primed and -infected wild-type mice. These results demonstrate that IL-17 is not required for development of severe pathology in response to infection with Borrelia burgdorferi, but may contribute to disease through an interaction with IFN-γ.

Tumor necrosis factor alpha, citrullination, and peptidylarginine deiminase 4 in lung and joint inflammation.

BACKGROUND: The relationship between lung and joint inflammation in rheumatoid arthritis is poorly understood. Lung inflammation with resultant protein citrullination may trigger anti-citrullinated protein antibodies, inflammation, and arthritis. Alternatively, lung and joint inflammation may be two manifestations of a single underlying pathology. The lung has increased citrullination and TNF-α levels are high in rheumatoid arthritis; however, it is unknown if TNF-α can induce lung protein citrullination. The citrullinating enzyme peptidylarginine deiminase 4 (PAD4) exacerbates TNF-α-induced arthritis, but a role for PAD4 in lung citrullination and TNF-α-induced lung inflammation has not been explored. Our aim was to use TNF-α-overexpressing mice to clarify the intersection of TNF-α, citrullination, PAD4, arthritis, and lung inflammation. METHODS: Lung protein citrullination in wild-type mice, mice that overexpress TNF-α systemically (TNF(+)), TNF(+)PAD4(+/+), and TNF(+)PAD4(-/-) mice was quantified by both gel electrophoresis using a citrulline probe and western blot. Hematoxylin and eosin (H&E)-stained lung sections from TNF(+)PAD4(+/+) and TNF(+)PAD4(-/-) mice were scored for lung inflammation. H&E-stained ankle joint sections from mice that overexpress TNF-α only in the lungs were assessed for arthritis. RESULTS: TNF(+) mice have increased lung protein citrullination. TNF(+)PAD4(-/-) mice do not have significantly reduced lung protein citrullination, but do have decreased lung inflammation compared to TNF(+)PAD4(+/+) mice. Mice that overexpress TNF-α only in the lungs do not develop arthritis. CONCLUSIONS: PAD4 exacerbates lung inflammation downstream of TNF-α without having a major role in generalized protein citrullination in inflamed lungs. Also, TNF-α-induced lung inflammation is not sufficient to drive murine arthritis.

Focal adhesion kinase is required for synovial fibroblast invasion, but not murine inflammatory arthritis.

INTRODUCTION: Synovial fibroblasts invade cartilage and bone, leading to joint destruction in rheumatoid arthritis. However, the mechanisms that regulate synovial fibroblast invasion are not well understood. Focal adhesion kinase (FAK) has been implicated in cellular invasion in several cell types, and FAK inhibitors are in clinical trials for cancer treatment. Little is known about the role of FAK in inflammatory arthritis, but, given its expression in synovial tissue, its known role in invasion in other cells and the potential clinical availability of FAK inhibitors, it is important to determine if FAK contributes to synovial fibroblast invasion and inflammatory arthritis. METHODS: After treatment with FAK inhibitors, invasiveness of human rheumatoid synovial fibroblasts was determined with Matrigel invasion chambers. Migration and focal matrix degradation, two components of cellular invasion, were assessed in FAK-inhibited rheumatoid synovial fibroblasts by transwell assay and microscopic examination of fluorescent gelatin degradation, respectively. Using mice with tumor necrosis factor α (TNFα)-induced arthritis in which fak could be inducibly deleted, invasion and migration by FAK-deficient murine arthritic synovial fibroblasts were determined as described above and arthritis was clinically and pathologically scored in FAK-deficient mice. RESULTS: Inhibition of FAK in human rheumatoid synovial fibroblasts impaired cellular invasion and migration. Focal matrix degradation occurred both centrally and at focal adhesions, the latter being a novel site for matrix degradation in synovial fibroblasts, but degradation was unaltered with FAK inhibitors. Loss of FAK reduced invasion in murine arthritic synovial fibroblasts, but not migration or TNFα-induced arthritis severity and joint erosions. CONCLUSIONS: FAK inhibitors reduce synovial fibroblast invasion and migration, but synovial fibroblast migration and TNFα-induced arthritis do not rely on FAK itself. Thus, inhibition of FAK alone is unlikely to be sufficient to treat inflammatory arthritis, but current drugs that inhibit FAK may inhibit multiple factors, which could increase their efficacy in rheumatoid arthritis.

Peptidylarginine deiminase 4 contributes to tumor necrosis factor α-induced inflammatory arthritis.

OBJECTIVE: Peptidylarginine deiminase 4 (PAD4) is a citrullinating enzyme that has multiple associations with inflammation. In rheumatoid arthritis, PAD4 and protein citrullination are increased in inflamed joints, and anti-citrullinated protein antibodies (ACPAs) form against citrullinated antigens are formed. ACPA immune complexes can deposit in the joint and induce the production of tumor necrosis factor α (TNFα), a critical inflammatory cytokine in the pathogenesis of rheumatoid arthritis. Further, in other settings, TNFα has been shown to induce PAD4 activity and modulate antibody formation. We undertook this study to investigate whether TNFα and PAD4 may synergistically exacerbate autoantibody production and inflammatory arthritis. METHODS: To determine whether TNFα and PAD4 augment autoantibody production and inflammatory arthritis, we first used a multiplex assay to determine whether mice with chronic inflammatory arthritis due to overexpression of TNFα develop autoantibodies against native and citrullinated antigens. With TNF(+) PAD4(+/+) and TNF(+) PAD4(-/-) mice, we then compared serum autoantibody levels by multiplex array, lymphocyte activation by flow cytometry, total serum IgG levels by enzyme-linked immunosorbent assay, arthritis by clinical and histologic scoring, and systemic inflammation using microfluidic devices. RESULTS: TNFα-overexpressing mice had increased levels of autoantibodies reactive against native and citrullinated antigens. PAD4(-/-) mice with TNFα-induced arthritis had lower levels of autoantibodies reactive against native and citrullinated antigens, decreased T cell activation and total IgG levels, and reduced inflammation and arthritis compared to PAD4(+/+) TNFα-overexpressing mice. CONCLUSION: PAD4 mediates autoantibody production and inflammatory arthritis downstream of TNFα.

Interleukin-10 (IL-10) inhibits Borrelia burgdorferi-induced IL-17 production and attenuates IL-17-mediated Lyme arthritis.

Previous studies have shown that cells and cytokines associated with interleukin-17 (IL-17)-driven inflammation are involved in the arthritic response to Borrelia burgdorferi infection. Here, we report that IL-17 is a contributing factor in the development of Lyme arthritis and show that its production and histopathological effects are regulated by interleukin-10 (IL-10). Spleen cells obtained from B. burgdorferi-infected, "arthritis-resistant" wild-type C57BL/6 mice produced low levels of IL-17 following stimulation with the spirochete. In contrast, spleen cells obtained from infected, IL-10-deficient C57BL/6 mice produced a significant amount of IL-17 following stimulation with B. burgdorferi. These mice developed significant arthritis, including erosion of the bones in the ankle joints. We further show that treatment with antibody to IL-17 partially inhibited the significant hind paw swelling and histopathological changes observed in B. burgdorferi-infected, IL-10-deficient mice. Taken together, these findings provide additional evidence of a role for IL-17 in Lyme arthritis and reveal an additional regulatory target of IL-10 following borrelial infection.

High-powered gas-cooled microwave ablation: shaft cooling creates an effective stick function without altering the ablation zone.

OBJECTIVE: The purpose of our study was to validate the ability of a new gas-cooled microwave device to secure antennas into tissue before ablation via shaft cooling and to verify that such cooling does not compromise the intended ablation. MATERIALS AND METHODS: The force required to extract several types of applicators from ex vivo bovine liver before and after ablation was measured. Six groups were compared: cooled needle and multitined radiofrequency electrodes, secured and unsecured cryoprobes, and gas-cooled microwave antennas (n = 6 each). Ablations were next created in in vivo porcine livers for 2 and 10 minutes (n = 6 each) using the gas-cooled microwave system at 140 W. Extraction force was again measured before and after ablation and compared between groups using analysis of variance with post hoc Student t tests. Histologic analysis of the ablation zone was performed to evaluate cellular necrosis along the antenna shaft. RESULTS: Ex vivo, the secured cryoprobe and microwave antenna required significantly more force to remove than unsecured radiofrequency, cryoprobe, and microwave applicators (p < 0.05, all comparisons). The multitined radiofrequency electrode and cooled radiofrequency electrode required significantly more force to remove after ablation than before ablation (p = 0.006 and 0.02, respectively). In vivo, the secured antenna required significantly more force to remove before ablation than after ablation at both 2 (p < 0.0001) and 10 minutes (p < 0.0001). There was no histologic evidence of cell preservation along the antenna shaft. CONCLUSION: The gas cooling used in this microwave device can effectively secure antennas into tissue without altering ablation shape or reducing the intended thermal damage.

Interleukin-35 enhances Lyme arthritis in Borrelia-vaccinated and -infected mice.

Interleukin-35 (IL-35) has been reported to inhibit the production of interleukin-17 (IL-17) as a means of preventing arthritis and other inflammatory diseases. We previously showed that treatment of Borrelia-vaccinated and -infected mice with anti-IL-17 antibody at the time of infection prevented the development of arthritis. The anti-IL-17 antibody-treated mice lacked the extensive tissue damage, such as bone and cartilage erosion, that occurred in the tibiotarsal joints of untreated Borrelia-vaccinated and -infected control mice. We hypothesized that IL-35 would reduce the severity of arthritis by suppressing the production of IL-17 in Borrelia-vaccinated and -infected mice. Here, we show that administration of recombinant IL-35 (rIL-35) to Borrelia-vaccinated and -infected mice augments the development of severe arthritis compared to the results seen with untreated control mice. Borrelia-vaccinated and -infected mice treated with rIL-35 had significantly (P < 0.05) greater hind paw swelling and histopathological changes from day 4 through day 10 than non-rIL-35-treated Borrelia-vaccinated and -infected mice. In addition, the treatment with IL-35 only slightly decreased the production of IL-17 in Borrelia-primed immune cells and did not prevent the development of borreliacidal antibody. Our data do not support a role for IL-35 as a potential therapeutic agent to reduce inflammation in Lyme arthritis.

Enlarging unilateral breast mass in an adolescent male: an unusual presentation of intraductal papilloma.

Breast masses presenting in adolescent boys are rare and are almost uniformly owing to gynecomastia. Although surgical referral for breast masses in adolescent boys is common, intervention is typically for cosmesis. We report the case of a 14-year-old boy who presented with an enlarging unilateral breast mass, which was found to be owing to an intraductal papilloma at the time of surgical excision.

Significant differences between the Borrelia-infection and Borrelia-vaccination and -infection models of Lyme arthritis in C3H/HeN mice.

The immunological events leading to the development of Lyme arthritis in humans are partially understood. Much of this information has been gained by studying the course of infection of naïve or vaccinated mice with Borrelia burgdorferi. However, the Borrelia-vaccination and -infection model has not been described using the organismal parameters commonly used in the widely accepted Borrelia-infection model. This is the first comparison between the Borrelia-infection and the Borrelia-vaccination and -infection models of arthritis. Borrelia-vaccinated and -infected C3H/HeN mice develop acute inflammation comparable to that of nonvaccinated, Borrelia-infected C3H/HeN mice. The duration and severity of arthritis in Borrelia-vaccinated and -infected mice was slightly increased compared with Borrelia-infected mice. Significantly, Borrelia-vaccinated and -infected C3H/HeN mice produce interleukin-17 (IL-17), while Borrelia-infected mice that had not been previously vaccinated do not. Neutralization of IL-17 in Borrelia-vaccinated and -infected C3H/HeN mice decreased the severity of arthritis, although not to the degree we observed previously in C57BL/6 mice. Collectively, these findings show that the Borrelia-vaccination and -infection model of Lyme arthritis incorporates elements of adaptive immunity that likely have relevance to human disease, but may not be observed in Borrelia-infected C3H/HeN mice.

Microthrombus formation may trigger lung injury after acute blood loss.

We showed previously that acute blood loss, without resuscitation, caused marked maldistribution of interalveolar perfusion. Because hemorrhage is a known risk factor for the development of lung injury, the goal of our present studies was to determine if there was a correlation between perfusion maldistribution and the subsequent development of lung injury after blood loss. Specifically, we wanted to know if the perfusion maldistribution might be due to microthrombus formation and/or leukocyte sequestration within the pulmonary microcirculation. We bled rats (30% blood loss) and harvested their lungs 45 min or 24 h later. Lungs were prepared for perfusion distribution analysis, Western blot analysis to measure whole-lung fibrinogen concentrations, and for immunohistochemistry to measure fibrin deposition and leukocyte deposition (CD16 fluorescence). Perfusion was significantly maldistributed at 45 min and 24 h (P < 0.05). At 45 min, whole-lung fibrinogen concentrations were less than half that in controls (P < 0.05), whereas numbers of fibrin microthrombi were 2.5-fold greater than control by 45 min (not statistically significant) and were 4.5-fold greater by 24 h (P = 0.01). Leukocyte deposition was two-fold greater than control by 45 min (not statistically significant) and was 4-fold greater by 24 h (P = 0.02). Fibrin-to-leukocyte nearest-neighbor distances remained unchanged (18.1 [SD, 1.1] µm) even as the numbers of both increased with time after blood loss. Our results suggest that soluble fibrinogen polymerized to insoluble fibrin within minutes after acute blood loss, which caused perfusion maldistribution and attracted leukocytes. The development of lung injury after blood loss may be a consequence of leukocyte chemoattraction to fibrin microthrombi that seem to form within minutes after blood loss.

Sebaceous cell carcinoma of the eyelid: a rapidly enlarging lesion with massive xanthogranulomatous inflammation.

A 76-year-old man presented atypically with a 4-week history of a rapidly enlarging ulcerated nodular lesion of the left upper eyelid that was found to be sebaceous cell carcinoma. Further investigation showed no metastatic disease, and Mohs surgery was performed to resect the tumor. Histopathologic analysis showed features diagnostic of sebaceous cell carcinoma. However, most of the mass consisted of xanthomatous granulomatous inflammatory reaction vastly out of proportion with the tumor burden. The patient was spared from orbital exenteration, and no evidence of recurrence was present 6 months after resection.

Discordant influence of Blastomyces dermatitidis yeast-phase-specific gene BYS1 on morphogenesis and virulence.

Blastomyces dermatitidis is a thermally induced dimorphic fungus capable of causing lung and systemic infections in immunocompetent animal hosts. With the publication of genomic sequences from three different strains of B. dermatitidis and the development of RNA interference as a gene-silencing tool, it has become possible to easily ascertain the virulence and morphological effects of knocking down the expression of candidate genes of interest. BYS1 (Blastomyces yeast-phase-specific 1), first identified by Burg and Smith, is expressed at high levels in yeast cells and is undetectable in mold. The deduced protein sequence of BYS1 has a putative signal sequence at its N terminus, opening the possibility that the BYS1-encoded protein is associated with the yeast cell wall. Herein, strains of B. dermatitidis with silenced expression of BYS1 were engineered and tested for morphology and virulence. The silenced strains produced rough-surfaced cultures on agar medium and demonstrated a propensity to form pseudohyphal cells on prolonged culture in vitro and in vivo, as measured in the mouse lung. Tests using a mouse model of blastomycosis with either yeast or spore inocula showed that the bys1-silenced strains were as virulent as control strains. Thus, although silencing of BYS1 alters morphology at 37 degrees C, it does not appear to impair the pathogenicity of B. dermatitidis.

One-year duration of immunity induced by vaccination with a canine Lyme disease bacterin.

Laboratory-reared beagles were vaccinated with a placebo or a bacterin comprised of Borrelia burgdorferi S-1-10 and ospA-negative/ospB-negative B. burgdorferi 50772 and challenged after 1 year with B. burgdorferi-infected Ixodes scapularis ticks. For the placebo recipients, spirochetes were recovered from 9 (60%) skin biopsy specimens collected after 1 month, and the organisms persisted in the skin thereafter. Ten (67%) dogs also developed joint infection (3 dogs), lameness or synovitis (7 dogs), or B. burgdorferi-specific antibodies (8 dogs). For the vaccine recipients, spirochetes were recovered from 6 (40%) skin biopsy specimens collected after 1 month. However, subsequent biopsy specimens were negative, and the dogs failed to develop joint infection (P = 0.224), lameness/synovitis (P = 0.006), or Lyme disease-specific antibody responses (P = 0.002). The bacterin provided a high level of protection for 1 year after immunization, and the addition of the OspC-producing B. burgdorferi 50772 provided enhanced protection.