兽研简要总结
猫利什曼病是一种由寄生虫引起、可感染猫的疾病。然而，人们对猫免疫系统如何应对这种感染知之甚少。在本研究中，我们使用猫巨噬细胞系来研究寄生虫亚马逊利什曼原虫如何与这些细胞相互作用。我们还检测了葡萄糖醛酸木甘露聚糖（GXM）的作用，这是一种由新型隐球菌产生的荚膜多糖，可改变免疫反应。我们发现猫巨噬细胞能够被亚马逊利什曼原虫感染，并随着时间部分控制寄生虫。然而，当GXM存在时，更多寄生虫在免疫细胞内存活。这种效应并非由巨噬细胞的损伤或死亡引起，而是由其免疫活性的变化所致。利什曼原虫抑制NO产生，GXM也减少了参与细胞天然防御感染的分子产生。这些发现展示了用于研究猫利什曼病的有用实验室模型，并提示真菌与寄生虫感染之间的相互作用可能影响疾病进展。
关键词：Fcwf-4猫巨噬细胞、利什曼原虫、免疫调节、纯化荚膜多糖、葡萄糖醛酸木甘露聚糖（GXM）
1. 引言
利什曼病是一组被忽视的人畜共患疾病，分布于全球90多个国家。巴西是皮肤型和内脏型利什曼病受影响最严重的国家之一，对人类和动物健康造成重大影响。犬被认定为利什曼原虫的主要城郊储存宿主。然而，越来越多的证据表明，家猫中利什曼原虫感染的流行率正在上升。在巴西，研究报告流行区猫感染率为6%至30%，在某些地区如圣保罗州阿拉萨图巴达到49%[1,2]。
过去40年间，全球约80项调查猫利什曼病（FeL）流行率的横断面研究报告平均血清阳性率为12%[3]。迄今为止，通过分子或同工酶分型方法，已在家猫中鉴定出属于利什曼原虫亚属的6种利什曼原虫和属于Viannia亚属的1种[4]。此外，已证实利什曼原虫在猫体表吸血时能够感染白蛉媒介[5]。总体而言，这些发现支持家猫可能作为利什曼原虫新兴储存宿主的假说。
在猫利什曼病的流行病学研究中也报道了与其他微生物的共感染，包括刚地弓形虫、孢子丝菌属、隐球菌属、猫冠状病毒，尤其是猫免疫缺陷病毒（FIV）和猫白血病病毒（FeLV）[6,7,8,9,10]。除共感染外，暴露于其他微生物来源的产物可能调节宿主易感性、免疫反应和疾病进展，可能促进猫利什曼原虫感染的建立和持续。
猫利什曼原虫感染的免疫学机制仍知之甚少，特别是涉及猫巨噬细胞及其与利什曼原虫相互作用的部分。最近，感染利什曼原虫（L. infantum）的猫单核细胞衍生巨噬细胞在体外感染期间显示Th1相关细胞因子表达上调[11]。同样，自然感染的猫血清IL-12和IFN-γ浓度高于未感染动物[12]。这些发现提示猫巨噬细胞可能对利什曼原虫感染产生独特的免疫反应。然而，调控巨噬细胞对利什曼原虫属反应的细胞和分子机制在很大程度上仍未探索，特别是在共感染或暴露于可能调节寄生虫存活和宿主炎症反应的微生物产物的背景下。
Fcwf-4细胞系来源于猫（Felis catus）胚胎巨噬细胞，是用于研究传染病[13,14,15,16]、药物重定位[17]、癌症治疗[18]和免疫反应[19,20]的成熟体外实验模型，因为该细胞系具有巨噬细胞样特性，如非特异性酯酶活性、吞噬能力、Fc受体和CD14表达[21,22]。在本研究中，我们提出将Fcwf-4猫巨噬细胞作为一种有意义的体外模型，用于研究巨噬细胞-利什曼原虫相互作用，并评估暴露于葡萄糖醛酸木甘露聚糖（GXM）——一种由新型隐球菌产生的免疫调节性荚膜多糖——是否影响猫巨噬细胞中的利什曼原虫感染。选择亚马逊利什曼原虫是因为该物种病例已在美洲报道[4,23]，并且是人类皮肤型和弥漫性皮肤型利什曼病的致病因子。
隐球菌病是猫的重要真菌病，也已在多种野生和外来动物物种中报道[24]。因此，研究GXM对利什曼原虫感染的影响可能有助于了解病原体来源分子如何调节巨噬细胞对寄生虫感染的易感性。这种实验方法可能有助于更好地理解猫利什曼病的细胞相互作用，特别是在暴露于微生物产物的背景下。
2. 材料与方法
2.1. 猫巨噬细胞系
猫巨噬细胞系 Fcwf-4 购自里约热内卢细胞库（BCRJ 编号 0334），培养于添加 10% 胎牛血清（FBS；Gibco，纽约，纽约州，美国）的 DMEM 培养基（Sigma-Aldrich，圣路易斯，密苏里州，美国）中，并维持于 37 °C/5% CO2 条件下。
2.2. 寄生虫
亚马逊利什曼原虫前鞭毛体（MHOM/BR/75/Josefa）维持于添加 10% 胎牛血清（Gibco）的 Schneider 昆虫培养基（Sigma）中，27 °C 培养。培养 4 天后，寄生虫用 PBS（磷酸盐缓冲盐水）洗涤，于 22 °C、2000× g 离心 10 分钟。细胞重悬于 PBS 中，并使用 Neubauer 计数板（Sigma-Aldrich，圣路易斯，密苏里州，美国）计数。
2.3. 荚膜 GXM 纯化
葡糖醛酸木甘露聚糖（GXM）从有荚膜的新型隐球菌菌株培养上清中分离，采用改编自先前描述方法的方案 [A, B]。简言之，真菌细胞在限定液体培养基中于 37 °C 连续振荡（100 rpm）培养 5 天。孵育后，于 4 °C、12,000× g 离心 1 小时去除细胞。无细胞上清中存在的荚膜 GXM 随后通过加入三倍体积冷乙醇沉淀回收。
GXM 进一步通过十六烷基三甲基溴化铵（CTAB）差异沉淀法纯化，如先前所述 [25]。将乙醇沉淀的多糖组分以 10 mg/mL 浓度溶解于 0.2 M NaCl 中。按每毫克多糖 3 mg 的比例逐渐加入 CTAB，随后加入 0.05% CTAB 溶液以促进 GXM 的选择性沉淀。收集所得沉淀 by 离心，并依次用含 2% 乙酸的乙醇和 90% 乙醇洗涤。然后将含 GXM 的沉淀溶解于 1 M NaCl 中，随后加入三倍体积乙醇诱导第二次沉淀步骤。离心后，沉淀再次用含 2% 乙酸的乙醇洗涤，随后用乙醇洗涤。最后，将纯化的 GXM 溶解于蒸馏水中并冻干。
为尽量减少潜在脂多糖（LPS）污染，将冻干的 GXM 制品溶解于无 LPS 水中，并使用经无 LPS 水平衡的多粘菌素 B-琼脂糖柱（Sigma-Aldrich，圣路易斯，密苏里州，美国）进行额外纯化步骤。GXM 用 12 mL 无 LPS 水洗脱，收集并在实验使用前冻干。
所有 GXM 制品在用于细胞实验前也均筛查了潜在支原体污染。仅支原体污染检测阴性的制品用于后续试验。
2.4. 感染利什曼原虫的猫巨噬细胞活力评估
将 Fcwf-4 猫巨噬细胞接种（2 × 105 个细胞/孔）于 96 孔板中，于 37 °C/5% CO2 孵育 2 小时后去除未贴壁细胞。随后细胞用或不用 100 µg/mL GXM 处理并孵育 24 小时。之后，细胞以 10 个寄生虫：1 个巨噬细胞的比例感染亚马逊利什曼原虫前鞭毛体，并于 37 °C/5% CO2 孵育 2 小时。然后洗涤细胞并继续孵育 24 和 48 小时。孵育后，细胞用 37 °C 的 PBS 洗涤，并与 50 µL 含 10% 吩嗪硫酸甲酯（PMS，500 µM）的 XTT 溶液（Sigma-Aldrich；0.001 g/mL）孵育。于 37 °C 作用 4 小时后，使用酶标仪在 450 nm 处测定吸光度。细胞活力（%）相对于未处理、未感染的对照细胞（100%）确定。10% TritonX-100 用作阴性对照。
2.5. 感染动力学及感染指数测定
将 Fcwf-4 猫巨噬细胞接种（2 × 105 个细胞/孔）于 24 孔板中的 13 mm 玻璃盖玻片上。于 37 °C/5% CO2 培养 2 小时后去除未贴壁细胞。细胞用 GXM [100 µg/mL] 处理 24 小时，然后感染亚马逊利什曼原虫前鞭毛体（10 个寄生虫：1 个巨噬细胞比例）2 小时。洗涤后，细胞孵育 2、24 和 48 小时。盖玻片采用 Giemsa 法染色。感染指数（% 感染巨噬细胞 × 每个巨噬细胞的寄生虫数）通过在光学显微镜（1000× 放大）下计数至少 200 个细胞确定。
2.6. 寄生虫负荷
将 Fcwf-4 猫巨噬细胞接种（2 × 105 个细胞/孔）于 24 孔板中。去除未贴壁细胞后，巨噬细胞用 GXM [100 ng/mL] 处理 24 小时，然后于 37 °C/5% CO2 感染亚马逊利什曼原虫前鞭毛体（10 个寄生虫：1 个巨噬细胞比例）2 小时。洗涤细胞并在 DMEM 培养基中孵育 24 小时。随后，洗涤细胞并加入 Schneider 昆虫培养基，于 27 °C 孵育。在孵育后第 3、5 和 7 天，使用 Neubauer 计数板计数上清中从细胞释放的寄生虫。
2.7. 一氧化氮（NO）检测
将Fcwf-4猫巨噬细胞（2×10^5个/孔）接种于96孔板中，在37 °C/5% CO2条件下用或不用100 µg/mL GXM处理24小时。随后用或不用亚马逊利什曼原虫（比例10:1）感染细胞。2小时后，洗涤培养物并加入含或不含LPS（400 ng/mL）的新鲜培养基孵育24小时。收集上清液并离心。将样品转移至新板，并制备亚硝酸钠标准曲线。加入Griess试剂（A液：5%磷酸中的1%磺胺；B液：0.1% N-(1-萘基)乙二胺；Sigma；A液与B液1:1混合）50 µL。15分钟后，使用酶标仪（Varioskan LUX，Thermo Fisher Scientific，Waltham，MA，USA）在540 nm处测定吸光度。
活性氧（ROS）检测
将Fcwf-4猫巨噬细胞（2×10^5个/孔）接种并按上述方法处理。感染并用或不用LPS（400 ng/mL）孵育24小时后，洗涤细胞并在37 °C避光条件下用CM-H2DCFDA探针（10 µM；invitrogen）孵育60分钟。使用荧光读数仪（Varioskan LUX）在激发波长493 nm和发射波长522 nm处测定荧光强度。
2.8. 统计分析
所有数据均使用GraphPad Prism 9.5软件进行分析。数据正态性采用D'Agostino–Pearson综合正态性检验评估。两组间比较采用Student's t检验。涉及三组或以上比较时采用单因素方差分析，涉及两个自变量的分析采用双因素方差分析。以* p < 0.05认为差异具有统计学意义。
3. 结果
为建立Fcwf-4猫巨噬细胞系作为亚马逊利什曼原虫感染的体外模型，我们首先评估了其与寄生虫相互作用并支持感染的能力。Fcwf-4巨噬细胞经荚膜多糖葡萄糖醛酸木甘露聚糖（GXM）预处理或未经处理，随后与亚马逊利什曼原虫前鞭毛体孵育2、24或48小时。测定感染指数，该指数综合考虑感染巨噬细胞的百分比及每个感染细胞内寄生虫的数量。
我们的结果表明，Fcwf-4巨噬细胞能够有效识别、结合并内化亚马逊利什曼原虫前鞭毛体，证实该猫源细胞系对寄生虫感染具有易感性。对每个巨噬细胞内寄生虫数量的分析显示，相互作用2小时后，未处理组与GXM处理组之间无显著差异，表明GXM预处理未影响初始的寄生虫摄取（图1A）。然而，感染24小时后，暴露于GXM的巨噬细胞显示出显著更高的寄生虫负荷，平均每个巨噬细胞含1.9个胞内寄生虫，而未处理细胞为每个巨噬细胞1.3个寄生虫。至48小时，尽管仍可检测到胞内寄生虫，但两组间每个巨噬细胞的寄生虫数量无统计学显著差异（图1A）。
[图：亚马逊利什曼原虫感染Fcwf-4猫巨噬细胞。Fcwf-4巨噬细胞用或不用GXM [100]
感染巨噬细胞的百分比呈现明显的时间模式。在最早的时间点（2小时），约62%的未处理巨噬细胞被感染，而GXM预处理导致感染细胞比例较低（49%），提示GXM可能短暂干扰初始的寄生虫-细胞相互作用（图1B）。24小时时，实验组间未检测到显著差异。相反，感染48小时后，GXM处理巨噬细胞的感染细胞比例（24%）显著高于未处理巨噬细胞（10%），表明GXM有利于感染随时间的维持或持续（图1B）。
当将两个参数整合为感染指数时，GXM预处理显著增强了Fcwf-4巨噬细胞对亚马逊利什曼原虫的易感性。与未处理对照相比，GXM处理巨噬细胞的感染指数在24小时时高2.9倍，在48小时时高3.04倍（图1C）。代表性光学显微镜图像证实了这些定量结果，显示GXM处理巨噬细胞内的寄生虫负荷明显高于未处理细胞（图2）。猫巨噬细胞内寄生虫负荷分析显示，GXM处理细胞释放的活寄生虫数量高于未处理猫巨噬细胞（图3）。
[图：亚马逊利什曼原虫感染的Fcwf-4猫巨噬细胞在2（A）、24（B）和48小时（C）的情况。GXM处理的亚马逊利什曼原虫感染]
[图：Fcwf-4猫巨噬细胞中寄生虫负荷的分析。猫巨噬细胞用或不用GXM [100 µg/mL] for ]
为排除寄生虫负荷增加源于GXM或寄生虫感染的细胞毒性效应这一可能性，在所有实验条件下评估了巨噬细胞活力。在整个实验期间细胞活力始终保持在较高水平，无论GXM处理或亚马逊利什曼原虫感染如何，孵育24小时和48小时后均有约98%的细胞保持存活（图3）。这些发现表明，GXM和寄生虫感染均未损害巨噬细胞活力，证明GXM处理细胞中观察到的感染增强不能归因于细胞存活的差异。
由于一氧化氮（NO）是参与控制胞内病原体的主要巨噬细胞效应分子，我们接下来研究了GXM是否调节感染亚马逊利什曼原虫的Fcwf-4巨噬细胞中NO的产生。巨噬细胞在存在或不存在GXM的条件下培养，感染或不感染亚马逊利什曼原虫，并刺激或不刺激脂多糖（LPS）。正如预期，LPS刺激诱导了Fcwf-4巨噬细胞的强烈活化，与未刺激的对照细胞相比，亚硝酸盐产生增加了3.1倍（图4）。单独GXM处理或单独亚马逊利什曼原虫感染均未显著改变相对于未处理对照的基础NO产生。然而，亚马逊利什曼原虫显著抑制了LPS刺激诱导的NO反应，使亚硝酸盐产生减少约3.47倍。（图4）。
[图：Fcwf-4猫巨噬细胞活力。Fcwf-4巨噬细胞处理或不处理葡萄糖醛酸木甘露聚糖（GXM；100 µg/mL）并感]
活性氧（ROS）是巨噬细胞抗菌反应的另一个关键组分，也进行了评估。与未刺激的巨噬细胞相比，LPS刺激显著增加了ROS生成约1.25倍，证实了Fcwf-4细胞对炎症活化的反应性（图5）。相反，GXM处理显著降低了基础ROS产生约1.3倍，表明这种隐球菌多糖对巨噬细胞氧化代谢发挥抑制作用。单独亚马逊利什曼原虫感染相对于未感染对照巨噬细胞未显著改变ROS产生。然而，在LPS刺激条件下，GXM预处理显著减弱了氧化爆发，与未处理的LPS刺激巨噬细胞相比，ROS产生减少约1.6倍（图5）。综上所述，我们的发现表明Fcwf-4猫巨噬细胞对亚马逊利什曼原虫感染易感，且该寄生虫主动调节巨噬细胞效应反应，特别是通过抑制LPS刺激后的NO产生。重要的是，暴露于葡萄糖醛酸木甘露聚糖（GXM），一种新型隐球菌荚膜的免疫调节多糖，进一步增强了利什曼原虫的存活，并在静息、LPS刺激和亚马逊利什曼原虫感染条件下抑制了ROS产生。这些发现揭示了真菌来源的免疫调节分子改变巨噬细胞抗菌反应并促进胞内寄生虫持续存在的潜在机制。更广泛地说，我们的结果突出了GXM介导的免疫调节在塑造宿主-病原体相互作用中的潜在相关性，并为病原体来源分子如何在其原始感染背景之外影响免疫反应提供了新见解。（图6）。
[图：经GXM处理和/或感染亚马逊利什曼原虫的Fcwf-4猫巨噬细胞的一氧化氮（NO）产生。Fcwf-4]
[图：经GXM处理和/或感染亚马逊利什曼原虫的Fcwf-4猫巨噬细胞的活性氧（ROS）产生]
4. 讨论
探讨猫巨噬细胞对利什曼原虫感染反应的研究仍然有限，限制了我们对这一物种宿主-寄生虫相互作用细胞机制的理解。基于Fcwf-4细胞系建立的实验模型提供了一个受控且可重复的系统，用于研究猫巨噬细胞-利什曼原虫相互作用的早期事件，并评估寄生虫持续存在和免疫调节的机制。Fcwf-4细胞此前已在多个实验背景中使用，包括传染病模型[13,14,15,16]和免疫反应研究[19,20]。因此，使用这种猫巨噬细胞系可能为研究宿主-寄生虫相互作用提供有价值的平台，同时在实验研究的初始阶段减少对动物实验的需求。
我们的结果表明，Fcwf-4细胞对亚马逊利什曼原虫感染易感，在所有评估时间点（2、24和48小时）均可检测到胞内寄生虫。有趣的是，感染指数随时间下降，提示Fcwf-4巨噬细胞能够部分控制寄生虫负荷，但并不能完全清除感染。这些发现与近期观察结果一致，即猫单核细胞来源巨噬细胞可支持利什曼原虫（L. infantum）（与猫感染最常相关的利什曼原虫虫种）以及主要与爬行动物相关的蜥蜴利什曼原虫感染[26]。同样，猫血单核细胞来源巨噬细胞已被证明可在体外支持利什曼原虫（L. infantum）感染长达72小时[11]。然而，在感染利什曼原虫（L. infantum）的单核细胞来源巨噬细胞中，并未报道在Fcwf-4细胞中观察到的亚马逊利什曼原虫感染减少。使用犬和鼠巨噬细胞系的研究表明，亚马逊利什曼原虫通常表现为早期存活快速下降（24小时约下降40%），而利什曼原虫（L. infantum）可表现出更高的相对持续存在能力，具体取决于宿主巨噬细胞来源[27]。对感染利什曼原虫（L. infantum）和亚马逊利什曼原虫的鼠腹腔巨噬细胞进行蛋白质组学分析，鉴定出差异丰富的蛋白质，这些蛋白质可影响感染进程[28]。这些差异至少部分可能反映了各项研究中使用的利什曼原虫虫种和巨噬细胞模型的独特生物学特征。尽管亚马逊利什曼原虫作为一种皮肤型虫种并非在猫中鉴定到的主要虫种，但新大陆已有亚马逊利什曼原虫、巴西利什曼原虫和墨西哥利什曼原虫病例报道[4,23,29]，使用这些虫种有助于理解巨噬细胞的物种特异性反应。总之，这些发现表明Fcwf-4巨噬细胞可支持胞内亚马逊利什曼原虫感染，同时保留随时间部分限制寄生虫复制或持续存在的能力。
猫隐球菌病是一种重要的全身性真菌感染，猫中已有隐球菌属和利什曼原虫共感染的病例报道[10,21]。葡萄糖醛酸木甘露聚糖（GXM）是新型隐球菌的主要荚膜多糖，具有明确的免疫调节特性，并可在不同实验模型中改变先天免疫反应[30,31,32]。在本研究中，与未处理的感染细胞相比，GXM处理增加了感染亚马逊利什曼原虫的Fcwf-4巨噬细胞的感染指数。近期在暴露于GXM后感染硕大利什曼原虫的鼠巨噬细胞中也报道了类似效应[31]，提示GXM促进利什曼原虫持续存在的能力可能跨不同巨噬细胞物种和利什曼原虫模型存在。
GXM的免疫调节活性与多种机制相关，包括诱导IL-10产生、调节先天免疫反应、通过Fcγ受体II（FcγRII）信号传导以及产生TGF-beta，这些机制可激活抑制性通路[30,32]。这些效应可能通过损害巨噬细胞抗菌功能，促成GXM处理的Fcwf-4巨噬细胞中观察到的感染指数增加。重要的是，感染指数增加与细胞活力降低无关。Fcwf-4细胞活力在所有实验条件下均得以维持，这与既往研究结果一致，即GXM在浓度高达300 µg/mL、暴露24小时后对鼠腹腔巨噬细胞无细胞毒性[31]。因此，GXM处理后观察到的感染指数增加不太可能归因于宿主细胞死亡，而是支持GXM介导的免疫调节在促进胞内寄生虫持续存在中的作用。
为探究可能有助于Fcwf-4巨噬细胞部分控制亚马逊利什曼原虫感染的机制，我们评估了活性氮和活性氧 species 的产生。Fcwf-4巨噬细胞在400 ng/mL LPS刺激后产生NO，尽管该浓度与常用于刺激鼠巨噬细胞系（如RAW 264.7）的浓度相比相对较高。值得注意的是，亚马逊利什曼原虫感染降低了LPS刺激的J774-G8鼠巨噬细胞的NO产生，而未降低iNOS mRNA表达，提示该寄生虫影响NO合成的转录后事件[33]。有必要进一步研究以评估猫巨噬细胞是否如此。此外，感染硕大利什曼原虫并经GXM处理的鼠腹腔巨噬细胞未表现出NO产生的显著增加[31]。在本研究中，亚马逊利什曼原虫感染降低了猫巨噬细胞的NO产生，而GXM处理的细胞未观察到相同效应。由于GXM在鼠模型中的免疫调节作用是在M1/M2极化背景下解读的[31]，不能假定相同机制甚至相同效应会发生在感染亚马逊利什曼原虫的猫巨噬细胞中。由于活性氮 species 有助于巨噬细胞介导的胞内病原体控制，NO产生的抑制可能形成有利于利什曼原虫持续存在的细胞环境。
ROS的产生显示出类似的免疫调节模式。LPS刺激增加了Fcwf-4巨噬细胞的ROS产生，而与未处理细胞相比，GXM处理降低了ROS水平。这一观察结果与犬DH82巨噬细胞中的发现一致，在相同刺激条件下，LPS和IFN-γ增加了ROS产生，而GXM显著降低了ROS水平[34]。值得注意的是，GXM还降低了未刺激Fcwf-4巨噬细胞的基础ROS产生，进一步支持其对巨噬细胞活化的广泛抑制作用。相比之下，与未刺激的巨噬细胞相比，单独亚马逊利什曼原虫感染并未显著改变ROS产生。同样，来自CBA小鼠的鼠源巨噬细胞显示，与硕大利什曼原虫相比，亚马逊利什曼原虫未诱导ROS产生[35]。近期证据表明，ROS产生不仅仅是终末抗菌机制，还参与早期炎症信号传导和代谢适应，从而影响细胞因子产生和抗菌反应[36]。因此，GXM诱导的ROS减少（包括在基础条件下）可能干扰早期巨噬细胞活化和代谢适应，可能有助于形成允许利什曼原虫持续存在的细胞内环境。这一可能性值得进一步研究。
猫巨噬细胞部分控制利什曼原虫感染的能力也可能与猫和犬之间观察到的易感性差异有关。猫通常被认为比犬对临床利什曼病易感性更低，尽管持续性寄生虫血症可能发生[3]。流行地区的比较研究表明，感染利什曼原虫（L. infantum）的猫和犬在免疫发病机制方面具有若干共同特征，尽管犬可能比猫产生更强的抗体反应和更明显的寄生虫特异性免疫反应[9]。在自然感染的猫中，已有报道与健康动物相比血清IL-12和IFN-γ水平升高[12]，而感染利什曼原虫（L. infantum）的猫血单核细胞衍生巨噬细胞表现出促炎细胞因子表达增加[11]。总之，这些观察结果与以下假说相符：猫巨噬细胞能够对利什曼原虫产生有效的（尽管可能不完全的）促炎反应。然而，该反应是否直接导致猫临床疾病频率或严重程度较低仍有待确定。重要的是，合并感染或暴露于免疫调节性微生物组分（如GXM）可能改变这种平衡，并可能增加对利什曼原虫持续存在的易感性（图7）。在流行地区，猫自然暴露于利什曼原虫属以及合并感染/免疫调节剂，而经过验证的猫巨噬细胞反应模型将填补特定的方法学空白，因为大多数体外利什曼原虫-巨噬细胞研究是在鼠源或人源系统中进行的。因此，合并感染或其他微生物分泌产物的调节效应可能是影响猫利什曼病结局的重要因素，值得进一步研究，包括使用原代巨噬细胞。
[图：GXM介导的猫巨噬细胞中亚马逊利什曼原虫感染和巨噬细胞氧化反应的调节。Fc]
5. 结论
总体而言，我们的研究结果确立了Fcwf-4猫巨噬细胞作为研究利什曼原虫感染早期细胞事件和剖析猫巨噬细胞中寄生虫持续存在机制的有用体外模型。Fcwf-4细胞支持亚马逊利什曼原虫前鞭毛体感染，同时保留随时间部分限制感染的能力。重要的是，暴露于GXM增加了感染指数而不损害巨噬细胞活力，并与NO和ROS产生减少相关。这些发现提供了实验证据，表明真菌来源的免疫调节分子可能影响猫巨噬细胞中利什曼原虫感染的结局，并揭示了一种潜在机制，即合并感染或暴露于其他微生物来源产物可能改变宿主-寄生虫相互作用。因此，Fcwf-4模型可能为未来旨在确定猫巨噬细胞活化、寄生虫持续存在以及利什曼原虫与其他病原体之间相互作用的分子通路研究提供有用平台。
作者贡献
概念化，L.H.P.-d.-S.、D.D.-R.和E.B.d.S.-J.；方法学，F.d.P.P.、A.C.F.、R.Q.d.S.、J.d.M.B.、E.B.d.S.-J.、C.G.F.-d.-L.、L.M.-P.和J.O.P.；验证，L.H.P.-d.-S.、D.D.-R.、F.d.P.P.和C.G.F.-d.-L.；形式分析，F.d.P.P.、D.D.-R.、E.B.d.S.-J.、J.d.M.B.和L.H.P.-d.-S.；资源，L.H.P.-d.-S.、D.D.-R.和C.G.F.-d.-L.；数据管理，L.H.P.-d.-S.、D.D.-R.和F.d.P.P.；写作——原稿准备，L.H.P.-d.-S.；写作——审阅和编辑，L.H.P.-d.-S.、D.D.-R.、F.d.P.P.、D.d.O.N.和C.G.F.-d.-L.。所有作者均已阅读并同意稿件的发表版本。
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资助声明
本研究由里约热内卢州卡洛斯·查加斯·菲略研究资助基金会资助：E-26/210.800/2025；国家科学技术发展委员会：408487/2025-9 以及巴西高等教育人员改进协调委员会（CAPES）—资助代码 001。
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Simple Summary
Feline leishmaniasis is a disease caused by a parasite that can infect cats. However, little is known about how the cat’s immune system responds to this infection. In this study, we used a macrophage feline lineage to investigate how the parasite Leishmania amazonensis interacts with these cells. We also examined the effects of glucuronoxylomannan (GXM), a capsular polysaccharide produced by the Cryptococcus neoformans, which can alter immune responses. We found that feline macrophages were able to become infected with L. amazonensis and partially control the parasite over time. However, when GXM was present, more parasites survived inside the immune cells. This effect was not caused by damage or death of the macrophages, but rather by changes in their immune activity. Leishmania inhibited NO production and GXM also reduced the production of molecules involved in the cellular natural defenses against infections. These findings show a useful laboratory model for studying feline leishmaniasis and suggest that interactions between fungal and parasitic infections may influence disease progression.
Keywords: Fcwf-4 feline macrophages, Leishmania, immunomodulation, purified capsular polysaccharide, glucuronoxylomannan (GXM)
1. Introduction
Leishmaniasis comprises a group of neglected zoonotic diseases distributed worldwide across more than 90 countries. Brazil is among the countries most affected by both cutaneous and visceral forms of the disease, with substantial impacts on human and animal health. Dogs are recognized as the main periurban reservoir of Leishmania parasites. Nevertheless, increasing evidence indicates a growing prevalence of Leishmania infection among domestic cats. In Brazil, studies have reported feline infection rates ranging from 6% to 30% in endemic areas, reaching 49% in some regions, such as Araçatuba, São Paulo State [1,2].
Over the past 40 years, approximately 80 cross-sectional studies investigating the prevalence of feline Leishmaniasis (FeL) worldwide have reported an average seroprevalence of 12% [3]. To date, six Leishmania species belonging to the subgenus Leishmania and one species belonging to the subgenus Viannia have been identified in domestic cats using molecular or isoenzyme-based typing methods [4]. Furthermore, the ability of Leishmania parasites to infect sand fly vectors during blood feeding on cats has been demonstrated [5]. Collectively, these findings support the hypothesis that domestic cats may act as an emerging reservoir host for Leishmania.
Coinfections with other microorganisms have also been reported in epidemiological studies of cats with leishmaniasis, including Toxoplasma gondii, Sporothrix spp., Cryptococcus spp., feline coronavirus, and, particularly, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) [6,7,8,9,10]. In addition to coinfections, exposure to products derived from other microorganisms may modulate host susceptibility, immune responses, and disease progression, potentially contributing to the establishment and persistence of Leishmania infection in cats.
The immunological mechanisms underlying Leishmania infection in cats remain poorly understood, particularly those involving feline macrophages and their interactions with Leishmania parasites. Recently, feline monocyte-derived macrophages infected with L. infantum were shown to upregulate the expression of Th1-associated cytokines during in vitro infection [11]. Similarly, naturally infected cats exhibited higher serum concentrations of IL-12 and IFN-γ than uninfected animals [12]. These findings suggest that feline macrophages may mount a distinct immune response to Leishmania infection. However, the cellular and molecular mechanisms governing macrophage responses to Leishmania spp. remain largely unexplored, particularly in the context of coinfections or exposure to microbial products that may modulate parasite survival and host inflammatory responses.
The Fcwf-4 cell line, derived from feline (Felis catus) embryonic macrophages, represents an established in vitro experimental model for investigating infectious diseases [13,14,15,16], drug repurposing [17], cancer therapy [18], and immune responses [19,20], since this cell line has macrophage-like properties such as nonspecific esterase activity, phagocytic capacity, Fc receptor and CD14 expression [21,22]. In the present study, we propose Fcwf-4 feline macrophages as a meaningful in vitro model to investigate macrophage–Leishmania interactions and to evaluate whether exposure to glucuronoxylomannan (GXM), an immunomodulatory capsular polysaccharide produced by Cryptococcus neoformans, influences Leishmania infection in feline macrophages. The use of L. amazonensis species was choice since cases of this species have been reported in Americas [4,23] and is the causative agent of cutaneous and diffuse cutaneous in humans.
Cryptococcosis is an important fungal disease in cats and has also been reported in a variety of wild and exotic animal species [24]. Therefore, investigating the effects of GXM on Leishmania infection may provide insights into how pathogen-derived molecules can modulate macrophage susceptibility to parasitic infection. This experimental approach may contribute to a better understanding of the cellular interactions underlying feline Leishmaniasis, particularly in the context of exposure to microbial products.
2. Materials and Methods
2.1. Feline Macrophages Lineage
The feline macrophage cell line Fcwf-4 was purchased from Banco de células do Rio de Janeiro (BCRJ code 0334) and cultured in DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, New York, NY, USA) and maintained at 37 °C/5% CO2.
2.2. Parasites
L. amazonensis promastigotes (MHOM/BR/75/Josefa) were maintained in Schneider’s insect medium (Sigma) supplemented with 10% fetal bovine serum (Gibco) at 27 °C. After 4 days of culture, parasites were washed in PBS (phosphate-buffered saline) by centrifugation at 2000× g for 10 min at 22 °C. Cells were resuspended in PBS and counted using a Neubauer chamber (Sigma-Aldrich, St. Louis, MO, USA).
2.3. Capsular GXM Purification
Glucuronoxylomannan (GXM) was isolated from the culture supernatant of encapsulated Cryptococcus neoformans strains using a protocol adapted from previously described methods [A, B]. Briefly, fungal cells were cultivated in a defined liquid medium at 37 °C under continuous agitation (100 rpm) for 5 days. Following incubation, cells were removed by centrifugation at 12,000× g for 1 h at 4 °C. The capsular GXM present in the cell-free supernatant were subsequently recovered by precipitation with three volumes of cold ethanol.
GXM was further purified by differential precipitation using cetyltrimethylammonium bromide (CTAB), as previously described [25]. The ethanol-precipitated polysaccharide fraction was dissolved in 0.2 M NaCl at a concentration of 10 mg/mL. CTAB was gradually added at a ratio of 3 mg per mg of polysaccharide, followed by the addition of 0.05% CTAB solution to promote selective precipitation of GXM. The resulting precipitate was collected by centrifugation and sequentially washed with 2% acetic acid in ethanol and 90% ethanol. The GXM-containing precipitate was then dissolved in 1 M NaCl, followed by the addition of three volumes of ethanol to induce a second precipitation step. After centrifugation, the precipitate was washed again with 2% acetic acid in ethanol and subsequently with ethanol. Finally, the purified GXM was dissolved in distilled water and lyophilized.
To minimize potential lipopolysaccharide (LPS) contamination, the lyophilized GXM preparations were dissolved in LPS-free water and subjected to an additional purification step using a Polymyxin B-agarose column (Sigma-Aldrich, St. Louis, MO, USA) equilibrated with LPS-free water. GXM was eluted with 12 mL of LPS-free water, collected, and lyophilized prior to experimental use.
All GXM preparations were also screened for potential Mycoplasma contamination before being used in cell-based experiments. Only preparations that tested negative for Mycoplasma contamination were used in subsequent assays.
2.4. Evaluation of Viability of Feline Macrophages Infected with Leishmania spp.
Fcwf-4 feline macrophages were seeded (2 × 105 cells/well) in 96-well plates, and non-adherent cells were removed after 2 h of incubation at 37 °C/5% CO2. Cells were then treated or not with 100 µg/mL GXM and incubated for 24 h. Subsequently, cells were infected with L. amazonensis promastigotes at a ratio of 10 parasites:1 macrophage and incubated at 37 °C/5% CO2 for 2 h. Cells were then washed and further incubated for 24 and 48 h. After incubation, cells were washed with PBS at 37 °C and incubated with 50 µL of XTT solution (Sigma-Aldrich; 0.001 g/mL) containing 10% phenazine methosulfate (PMS, 500 µM). After 4 h at 37 °C, absorbance was measured at 450 nm using a microplate reader. Cell viability (%) was determined relative to untreated, uninfected control cells (100%). TritonX-100 10% was used as negative control.
2.5. Infection Kinetics and Determination of Infection Index
Fcwf-4 feline macrophages were plated (2 × 105 cells/well) on 13 mm glass coverslips in 24-well plates. Non-adherent cells were removed after 2 h at 37 °C/5% CO2. Cells were treated with GXM [100 µg/mL] for 24 h and then infected with L. amazonensis promastigotes (10 parasites:1 macrophage ratio) for 2 h. After washing, cells were incubated for 2, 24, and 48 h. Coverslips were stained using the Giemsa method. The infection index (% infected macrophages × number of parasites per macrophage) was determined by counting at least 200 cells under light microscopy (1000× magnification).
2.6. Parasite Load
Fcwf-4 feline macrophages were plated (2 × 105 cells/well) in 24-well plates. After removal of non-adherent cells, macrophages were treated with GXM [100 ng/mL] for 24 h and then infected with L. amazonensis promastigotes (10 p:1 m ratio) for 2 h at 37 °C/5% CO2. Cells were washed and incubated in DMEM medium for 24 h. Subsequently, cells were washed and Schneider’s insect medium was added, followed by incubation at 27 °C. Released parasites from the cells were counted in the supernatant using a Neubauer chamber on days 3, 5, and 7 post incubation.
2.7. Nitric Oxide (NO) Detection
Fcwf-4 feline macrophages (2 × 105 cells/well) were seeded in 96-well plates and treated or not with 100 µg/mL GXM for 24 h at 37 °C/5% CO2. Cells were then infected or not with L. amazonensis (10:1 ratio). After 2 h, cultures were washed and incubated with fresh medium with or without LPS (400 ng/mL) for 24 h. Supernatants were collected and centrifuged. Samples were transferred to a new plate, and a sodium nitrite standard curve was prepared. Griess reagent (1:1 mixture of solution A: 1% sulfanilamide in 5% phosphoric acid; solution B: 0.1% N-(1-naphthyl)ethylenediamine; Sigma) was added (50 µL). After 15 min, absorbance was measured at 540 nm using a microplate reader (Varioskan LUX, Thermo Fisher Scientific, Waltham, MA, USA).
Reactive Oxygen Species (ROS) Detection
Fcwf-4 feline macrophages (2 × 105 cells/well) were plated and treated as described above. After infection and incubation with or without LPS (400 ng/mL) for 24 h, cells were washed and incubated with CM-H2DCFDA probe (10 µM; invitrogen) for 60 min at 37 °C in the dark. Fluorescence intensity was measured using a fluorescence reader (Varioskan LUX) at excitation 493 nm and emission 522 nm.
2.8. Statistical Analysis
All data were analyzed using GraphPad Prism 9.5 software. Data normality was assessed using the D’Agostino–Pearson omnibus normality test. Comparisons between two groups were performed using Student’s t-test. For comparisons involving three or more groups, one-way ANOVA was used, and for analyses involving two independent variables, two-way ANOVA was applied. Differences were considered statistically significant at * p < 0.05.
3. Results
To establish the Fcwf-4 feline macrophage cell line as an in vitro model for Leishmania amazonensis infection, we first evaluated its ability to interact with and support parasite infection. Fcwf-4 macrophages were pretreated with the capsular polysaccharide glucuronoxylomannan (GXM) or left untreated before incubation with L. amazonensis promastigotes for 2, 24, or 48 h. Infection index, was determined, considering both the percentage of infected macrophages and the number of intracellular parasites per infected cell.
Our results demonstrated that Fcwf-4 macrophages efficiently recognized, bound, and internalized L. amazonensis promastigotes, confirming that this feline cell line is permissive to parasite infection. Analysis of the parasite per macrophage revealed no significant differences between untreated and GXM-treated macrophages after 2 h of interaction, indicating that GXM pretreatment did not influence the initial parasite uptake (Figure 1A). However, after 24 h of infection, macrophages exposed to GXM displayed a significantly greater parasite burden, with an average of 1.9 intracellular parasites per macrophage compared with 1.3 parasites per macrophage in untreated cells. By 48 h, although intracellular parasites remained detectable, no statistically significant differences were observed in the number of parasites per macrophage between the two groups (Figure 1A).
[图：Infection of Fcwf-4 feline macrophages with Leishmania amazonensis. Fcwf-4 macrophages were treated or not with GXM [100]
The percentage of infected macrophages followed a distinct temporal pattern. At the earliest time point (2 h), approximately 62% of untreated macrophages were infected, whereas GXM pretreatment resulted in a lower proportion of infected cells (49%), suggesting that GXM may transiently interfere with the initial parasite-cell interaction (Figure 1B). At 24 h, no significant differences were detected between the experimental groups. In contrast, after 48 h of infection, GXM-treated macrophages exhibited a significantly higher proportion of infected cells (24%) than untreated macrophages (10%), indicating that GXM favors the maintenance or persistence of infection over time (Figure 1B).
When both parameters were integrated into the infection index, GXM pretreatment markedly enhanced the susceptibility of Fcwf-4 macrophages to L. amazonensis. The infection index was 2.9-fold higher at 24 h and 3.04-fold higher at 48 h in GXM-treated macrophages compared with untreated controls (Figure 1C). Representative optical microscopy images corroborated these quantitative findings, showing a visibly greater intracellular parasite load in GXM-treated macrophages than in untreated cells (Figure 2). The parasite load analyses inside feline macrophages showed that the number of viable parasites released from GXM-treated cells were higher compared to untreated feline macrophages (Figure 3).
[图：L. amazonensis infected-Fcwf-4 feline macrophages at 2 ( A ), 24 ( B ) and 48 h ( C ). GXM treated- L. amazonenis infect]
[图：Analysis of parasite load in Fcwf-4 feline macrophages. Feline macrophages were treated or not with GXM [100 µg/mL] for ]
To exclude the possibility that the increased parasite burden resulted from cytotoxic effects of GXM or parasite infection, macrophage viability was evaluated under all experimental conditions. Cell viability remained consistently high throughout the experimental period, with approximately 98% of cells remaining viable after both 24 and 48 h of incubation, regardless of GXM treatment or L. amazonensis infection (Figure 3). These findings indicate that neither GXM nor parasite infection compromised macrophage viability, demonstrating that the enhanced infection observed in GXM-treated cells cannot be attributed to differences in cell survival.
Because nitric oxide (NO) is a major macrophage effector molecule involved in the control of intracellular pathogens, we next investigated whether GXM modulated NO production in Fcwf-4 macrophages infected with L. amazonensis. Macrophages were cultured in the presence or absence of GXM, infected or not with L. amazonensis, and stimulated or not with lipopolysaccharide (LPS). As expected, LPS stimulation induced a robust activation of Fcwf-4 macrophages, resulting in a 3.1-fold increase in nitrite production compared with unstimulated control cells (Figure 4). Neither GXM treatment alone nor L. amazonensis infection alone significantly altered basal NO production relative to untreated controls. However, L. amazonensis markedly suppressed the NO response induced by LPS stimulation, reducing nitrite production by approximately 3.47-fold. (Figure 4).
[图：Fcwf-4 feline macrophages Viability. Fcwf-4 macrophages were treated or not glucuronoxylomannan (GXM; 100 µg/mL) and inc]
Reactive oxygen species (ROS), another critical component of the macrophage antimicrobial response, were also evaluated. LPS stimulation significantly increased ROS generation by approximately 1.25-fold compared with unstimulated macrophages, confirming the responsiveness of Fcwf-4 cells to inflammatory activation (Figure 5). In contrast, GXM treatment significantly reduced basal ROS production by approximately 1.3-fold, indicating that this cryptococcal polysaccharide exerts an inhibitory effect on macrophage oxidative metabolism. Infection with L. amazonensis alone did not significantly alter ROS production relative to uninfected control macrophages. Nevertheless, under LPS-stimulated conditions, GXM pretreatment significantly attenuated the oxidative burst, reducing ROS production by approximately 1.6-fold compared with untreated LPS-stimulated macrophages (Figure 5). Taking together, our findings demonstrate that Fcwf-4 feline macrophages are permissive to Leishmania amazonensis infection and that the parasite actively modulates macrophage effector responses, particularly by suppressing nitric oxide (NO) production following LPS stimulation. Importantly, exposure to glucuronoxylomannan (GXM), an immunomodulatory polysaccharide of the C neoformans capsule, further enhanced Leishmania survival and inhibited reactive oxygen species (ROS) production under resting, LPS-stimulated, and L. amazonensis-infected conditions. These findings reveal a potential mechanism by which fungal-derived immunomodulatory molecules can alter macrophage antimicrobial responses and promote the persistence of an intracellular parasite. More broadly, our results highlight the potential relevance of GXM-mediated immunomodulation in shaping host–pathogen interactions and provide new insights into how pathogen-derived molecules may influence immune responses beyond their original infectious context. (Figure 6).
[图：Nitric Oxide (NO) production by Fcwf-4 feline macrophages treated with GXM and/or infected with L. amazonensis . Fcwf-4 ]
[图：Reactive species of oxygen (ROS) production by Fcwf-4 feline macrophages treated with GXM and/or infected with L. amazon]
4. Discussion
Studies addressing the feline macrophage response to Leishmania infection remain limited, restricting our understanding of the cellular mechanisms underlying host–parasite interactions in this species. The establishment of an experimental model based on the Fcwf-4 cell line provides a controlled and reproducible system to investigate early events in feline macrophage–Leishmania interactions and to assess mechanisms underlying parasite persistence and immune modulation. Fcwf-4 cells have previously been used in several experimental contexts, including infectious disease models [13,14,15,16], and studies of immune responses [19,20]. Thus, the use of this feline macrophage cell line may provide a valuable platform for investigating host–parasite interactions while reducing the need for animal experimentation during the initial stages of experimental studies.
Our results demonstrate that Fcwf-4 cells are susceptible to infection with L. amazonensis, with intracellular parasites detected at all evaluated time points (2, 24, and 48 h). Interestingly, the infection index decreased over time, suggesting that Fcwf-4 macrophages can partially control parasite burden without complete eliminating the infection. These findings are consistent with recent observations showing that feline monocyte-derived macrophages can sustain infection with L. infantum, the Leishmania species most frequently associated with feline infections, as well as with L. tarentolae, a species primarily associated with reptiles [26]. Similarly, feline blood monocyte-derived macrophages have been shown to support L. infantum infection in vitro for up to 72 h [11]. However, the reduction in L. amazonensis infection observed in Fcwf-4 cells was not reported in L. infantum-infected monocyte-derived macrophages. Studies using canine and murine macrophage cell lines show that L. amazonensis often exhibits rapid early survival reduction (around 40% at 24 h), whereas L. infantum can display higher relative persistence depending on the host macrophage origin [27]. Proteomic analysis in murine peritoneal macrophages infected with L. infantum and L. amazonensis identified differential protein abundant, which can affect infection course [28]. These differences may reflect, at least in part, the distinct biological characteristics of the Leishmania species and macrophage models used in each study. Although L. amazonensis, a cutaneous species, is not the main species identified in cats, cases of L. amazonesnsis, L. braziliensis and L. mexicana have been reported in New World [4,23,29], the use of these species can contribute to understand macrophages species-specific response. Collectively, these findings indicate that Fcwf-4 macrophages can support intracellular L. amazonensis infection while retaining the capacity to partially restrict parasite replication or persistence over time.
Feline cryptococcosis is an important systemic fungal infection, and cases of Cryptococcus spp. and Leishmania co-infection have been reported in cats [10,21]. Glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, has well-established immunomodulatory properties and can alter innate immune responses in different experimental models [30,31,32]. In the present study, GXM treatment increased the infection index in L. amazonensis-infected Fcwf-4 macrophages compared with untreated infected cells. A similar effect was recently reported in murine macrophages infected with L. major following GXM exposure [31], suggesting that the ability of GXM to promote Leishmania persistence may extend across different macrophage species and Leishmania models.
The immunomodulatory activity of GXM has been associated with several mechanisms, including induction of IL-10 production, modulation of innate immune responses, signaling through Fcγ receptor II (FcγRII), and TGF-beta production, which can activate inhibitory pathways [30,32]. These effects may contribute to the increased infection index observed in GXM-treated Fcwf-4 macrophages by impairing macrophage antimicrobial functions. Importantly, the increased infection index was not associated with reduced cell viability. Fcwf-4 cell viability was maintained under all experimental conditions, consistent with previous findings showing that GXM is not cytotoxic to murine peritoneal macrophages at concentrations of up to 300 µg/mL following 24 h of exposure [31]. Therefore, the increased infection index observed following GXM treatment is unlikely to be attributable to host–cell death and instead supports a role for GXM-mediated immunomodulation in facilitating intracellular parasite persistence.
To investigate mechanisms potentially contributing to the partial control of L. amazonensis infection by Fcwf-4 macrophages, we evaluated the production of reactive nitrogen and oxygen species. Fcwf-4 macrophages produced NO following stimulation with 400 ng/mL LPS, although this concentration is relatively high compared with concentrations commonly used to stimulate murine macrophage cell lines such as RAW 264.7. Notably, L. amazonensis infection reduced NO production in LPS-stimulated J774-G8murine macrophages without decreased iNOS mRNA expression, suggesting that the parasite affects post-transcriptional events of NO synthesis [33]. It is necessary further investigation to evaluate if it is the case of feline macrophages. Furthermore, murine peritoneal macrophages infected with L. major and treated with GXM did not exhibit a significant increase in NO production [31]. In the present study, L. amazonensis infection reduced NO production in feline macrophages, the same effect was not observed by GXM treated cells. Since GXM immunomodulatory action in the murine model was interpreted in the context of M1/M2 polarization [31] it cannot be assumed that the same mechanism, or even the same effect, will occur in feline macrophages infected with L. amazonensis. Because reactive nitrogen species contribute to macrophage-mediated control of intracellular pathogens, the suppression of NO production may create a cellular environment that favors Leishmania persistence.
ROS production showed a similar pattern of immunomodulation. LPS stimulation increased ROS production by Fcwf-4 macrophages, whereas GXM treatment reduced ROS levels compared with untreated cells. This observation is consistent with findings in canine DH82 macrophages, in which LPS and IFN-γ increased ROS production, while GXM significantly reduced ROS levels under the same stimulatory conditions [34]. Notably, GXM also reduced basal ROS production in unstimulated Fcwf-4 macrophages, further supporting its broad inhibitory effect on macrophage activation. In contrast, L. amazonensis infection alone did not significantly alter ROS production compared with unstimulated macrophages. Similarly in murine macrophages from CBA mice showed that L. amazonensis did not induce ROS production compared to L. major [35]. Recent evidence indicates that ROS production is not merely a terminal antimicrobial mechanism but also participates in early inflammatory signaling and metabolic adaptation, thereby influencing cytokine production and antimicrobial responses [36]. Therefore, the reduction in ROS induced by GXM, including under basal conditions, may interfere with early macrophage activation and metabolic adaptation, potentially contributing to an intracellular environment permissive for Leishmania persistence. This possibility warrants further investigation.
The ability of feline macrophages to partially control Leishmania infection may also be relevant to the differences in susceptibility observed between cats and dogs. Cats are generally considered less susceptible to clinical Leishmaniasis than dogs, although persistent parasitemia can occur [3]. Comparative studies in endemic areas have suggested that cats and dogs infected with L. infantum share several features of immunopathogenesis, although dogs may develop stronger antibody responses and more pronounced parasite-specific immune responses than cats [9]. In naturally infected cats, increased serum levels of IL-12 and IFN-γ have been reported compared with healthy animals [12], while feline blood monocyte-derived macrophages infected with L. infantum exhibit increased expression of proinflammatory cytokines [11]. Together, these observations are compatible with the hypothesis that feline macrophages can mount an effective, although potentially incomplete, pro-inflammatory response to Leishmania. However, whether this response directly contributes to the lower frequency or severity of clinical disease in cats remains to be established. Importantly, concomitant infections or exposure to immunomodulatory microbial components such as GXM could alter this balance and potentially increase susceptibility to Leishmania persistence (Figure 7). Cats are naturally exposed to both Leishmania spp. and to co-infections/immunomodulatory agents in endemic areas, and a validated a model of feline macrophage response would fill a specific methodological gap, since most in vitro Leishmania–macrophage has been done in murine or human systems. Thus, coinfection or the modulatory effects of products secreted by other microorganisms may represent an important factor influencing the outcome of feline leishmaniasis and deserves further investigation, including using primary macrophages.
[图：GXM-mediated modulation of Leishmania amazonensis infection and macrophage oxidative responses in feline macrophages. Fc]
5. Conclusions
Overall, our findings establish Fcwf-4 feline macrophages as a useful in vitro model for investigating early cellular events during Leishmania infection and for dissecting mechanisms of parasite persistence in feline macrophages. Fcwf-4 cells supported infection with L. amazonensis promastigotes while retaining the capacity to partially restrict the infection over time. Importantly, exposure to GXM increased the infection index without compromising macrophage viability and was associated with reduced NO and ROS production. These findings provide experimental evidence that fungal-derived immunomodulatory molecules may influence the outcome of Leishmania infection in feline macrophages and highlight a potential mechanism by which co-infections or exposure to products derived from other microorganisms could modify host–parasite interactions. The Fcwf-4 model may therefore provide a useful platform for future studies aimed at defining the molecular pathways in feline macrophage activation, parasite persistence, and the interplay between Leishmania and other pathogens.
Author Contributions
Conceptualization, L.H.P.-d.-S., D.D.-R. and E.B.d.S.-J.; methodology, F.d.P.P., A.C.F., R.Q.d.S., J.d.M.B., E.B.d.S.-J., C.G.F.-d.-L., L.M.-P. and J.O.P.; validation, L.H.P.-d.-S., D.D.-R., F.d.P.P. and C.G.F.-d.-L.; formal analysis, F.d.P.P., D.D.-R., E.B.d.S.-J., J.d.M.B. and L.H.P.-d.-S.; resources, L.H.P.-d.-S., D.D.-R. and C.G.F.-d.-L.; data curation, L.H.P.-d.-S., D.D.-R. and F.d.P.P.; writing—original draft preparation, L.H.P.-d.-S.; writing—review and editing, L.H.P.-d.-S., D.D.-R., F.d.P.P., D.d.O.N. and C.G.F.-d.-L. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding Statement
This research was funded by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro: E-26/210.800/2025; Conselho Nacional de Desenvolvimento Científico e Tecnológico: 408487/2025-9 and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001.
Footnotes
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References
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Associated Data
Data Availability Statement
The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.