Immunosuppressive Ability of Trichinella spiralis Adults Can Ameliorate Type 2 Inflammation in a Murine Allergy Model

Abstract Background There is an increase in the global incidence of allergies. The hygiene hypothesis and the old friend hypothesis reveal that helminths are associated with the prevalence of allergic diseases. The therapeutic potential of Trichinella spiralis is recognized; however, the stage at which it exerts its immunomodulatory effect is unclear. Methods We evaluated the differentiation of bone marrow–derived macrophages stimulated with T spiralis excretory-secretory products. Based on an ovalbumin-induced murine model, T spiralis was introduced during 3 allergy phases. Cytokine levels and immune cell subsets in the lung, spleen, and peritoneal cavity were assessed. Results We found that T spiralis infection reduced lung inflammation, increased anti-inflammatory cytokines, and decreased Th2 cytokines and alarms. Recruitment of eosinophils, CD11b+ dendritic cells, and interstitial macrophages to the lung was significantly suppressed, whereas Treg cells and alternatively activated macrophages increased in T spiralis infection groups vs the ovalbumin group. Notably, when T spiralis was infected prior to ovalbumin challenge, intestinal adults promoted proportions of CD103+ dendritic cells and alveolar macrophages. Conclusions T spiralis strongly suppressed type 2 inflammation, and adults maintained lung immune homeostasis.

Allergies, typical immune-mediated inflammatory diseases, are on the rise globally [1].The immunoregulatory potential of helminths has substantial therapeutic potential in immunemediated inflammatory diseases, such as inflammatory bowel diseases and allergies [2][3][4][5].However, the relationship between the Th2-type immune response and the immunosuppressive response induced by helminths such as Trichinella spiralis makes it challenging to discern their immunoregulatory effects on inflammatory diseases.T spiralis has a unique life cycle, completing parasitism within the same host across various stages-intestinal, migration, and muscular.Infective muscular larvae (ML) undergo 4 molts at 31 hours postinfection [6], developing into adults that reach sexual maturity and fertilization within 3 days in the intestinal tract [7].Female adults produce newborn larvae (NBL) after 5 days that eventually parasitize muscle cells via the circulatory system [8].Adult worms in the intestines are eventually eliminated [9].
The innate and adaptive immune systems of the host resist T spiralis through killing, weeping (intestinal mucus secretion), and sweeping (enhanced intestinal peristalsis) mechanisms.Macrophages and neutrophils contribute to killing T spiralis [10], whereas innate lymphoid cells 2, Th2 cells, and intestinal epithelial cells induce a strong type 2 immune response to eliminate T spiralis from the intestine [11].However, T spiralis has developed a sophisticated immune evasion mechanism for long-term coexistence with hosts [12].Because of the unique life cycle of T spiralis, the effects of its 3 life stages on the host immune response are different.Furthermore, the 3 stages of T spiralis cannot be entirely discerned, making it difficult to distinguish the immunomodulatory effects of different developmental stages.The ownership of the regulatory capacity of Th2-type immune responses and immunosuppressive responses remains elusive.
We investigated the regulatory effect of excretory-secretory products (ESPs) at different stages on bone marrow-derived macrophages (BMDMs) in vitro [13,14].Based on the immunoregulation of ESPs, our study examined the anti-inflammatory The Journal of Infectious Diseases M A J O R A R T I C L E effect and immunoregulatory ownership of adults at 3 stages of allergic inflammation: the sensitization, effector, and treatment stages.We assessed changes in immune cells and cytokines in an ovalbumin (OVA)-induced model.The inhibitory effect of adults on allergic inflammation was stronger in the sensitization and effector phases than in the treatment phase.Our findings suggest that T spiralis adults induce an immunosuppressive response to reduce OVA-induced type 2 inflammation.

Animals and Parasites
Female C56BL/6 mice and Wistar rats aged 6 to 8 weeks were procured from the Jilin University Experimental Animal Center.All animal experiments adhered to the guidelines for the care and use of laboratory animals [15] and were approved by the Ethical Committee of Jilin University, affiliated with the Provincial Animal Health Committee (ethical clearance KT202202140).T spiralis (ISS534) was obtained from the serial passage of rats in our laboratory.

Asthma Model and Infection With T spiralis
Mice were initially sensitized with 20-μg OVA and 50-μL Imject Alum adjuvant (Thermo Fisher Scientific) in a 100-μL volume on days 0, 7, and 14.Control groups consisted of mice administered with either saline or alum adjuvant.Mice were subsequently exposed to aerosolized 1% OVA for 30 minutes via the airway on days 28, 29, and 30.The mice were humanely euthanized on day 37.In alignment with the infection timing, the group infected with T spiralis larvae was stratified into 4 subgroups corresponding to days −3, 15, 25, and 31.

Culture and Induction of BMDMs
Bone marrow cells were obtained from the isolated leg bones.The cells were treated with red cell lysate, cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 25 ng/mL of recombinant murine macrophage colony-stimulating factor, and incubated in an environment at 37 °C and with 5% CO 2 .The same medium was replaced 2 days later, and ESPs (at 20-and 50-μg concentrations) were added for 24 hours on day 4. Mature BMDMs were collected on day 5 for flow cytometry analysis, and the supernatant was collected to assess cytokine levels.

Statistical Analysis
All data were analyzed with Prism software (version 5; GraphPad), and the results are presented as mean and SD.To assess differences among groups, 1-and 2-way analyses of variance were employed.Significance levels were denoted as *P < .05,**P < .01,and ***P < .001.

Differentiation of BMDMs Induced by ESPs of T spiralis
The proportion of F4/80 + BMDMs exceeded 98% (Supplementary Figure 1A), indicating the high purity and reactivity of the BMDMs.Stimulation with rmIL-4 resulted in an increased proportion of CD206 + macrophages (Figure 1A and 1B), confirming the reactivity of BMDMs.When stimulated with 20 μg of ESPs, the CD206 expression was upregulated by ESPs (AD3 and ML), whereas ESPs (30 hours and AD6) did not exhibit the same effect.As the dose of ESPs increased to 50 μg, CD206 expression was consistently upregulated, except in the AD6 group.The cytokine profiles secreted by BMDMs varied in response to different ESPs.All ESPs led to upregulated levels of interleukins 12p70 and 1β (IL-12p70 and IL-1β), with the AD6 group (20 and 50 μg) showing particularly high levels.Additionally, ESPs induced elevated levels of anti-inflammatory cytokines (interleukin 10 [IL-10] and transforming growth factor β1 [TGF-β1]), particularly in the 3 groups (30 hours, AD3, and ML; Figure 1C).Among these, ESPs (AD3) induced the highest levels of anti-inflammatory cytokines, consistent with CD206 expression, indicating that ESPs (AD3) possess a strong ability to induce the alternative activation of macrophages.

T spiralis Adults Alleviate Allergic Inflammation in the Lungs
Considering the regulatory effect of ESPs on macrophages, we artificially infected mice with T spiralis and observed immunoregulatory effects at 3 stages: sensitization (day −3 and 15 of infection), challenge (day 25 of infection), and treatment (day −3; Figure 2A).The infection efficiency of NBL was determined by quantifying the ML (Supplementary Figure 2).The OVA sensitization and challenge group (OVA group) exhibited more inflammatory cell infiltration, mucus production, and bronchiolar wall proliferation than the saline and adjuvant control groups (Figure 2B and 2C).In the T spiralis infection group at day −3, the ML presence affected the subsequent OVA challenge stage.Therefore, mice were infected with NBL via the tail vein on day 15 as a control.Adults significantly reduced inflammatory cell infiltration, goblet cell metaplasia, and mucus production in lung tissue during the sensitization, challenge, and treatment stages.NBL infection was less effective in suppressing allergic inflammation than adult infection at day −3.Cell counts in bronchoalveolar lavage fluid (BALF) included various immune cells, such as macrophages, neutrophils, eosinophils, and lymphocytes.The results showed that adults reduced total cell counts and macrophage and eosinophil infiltration induced by OVA, particularly on days −3 and 25 of the infection groups (Figure 2D).

T spiralis Adults Reduce OVA-Specific Immunoglobulin
We evaluated the effect of T spiralis on OVA-specific immunoglobulin in serum (Figure 3A).OVA-immunoglobulin G (IgG)  Trichinella spiralis mitigates allergic inflammation in the lungs.A, Experimental design.Mice underwent sensitization with OVA/alum via intraperitoneal injection on days 0, 7, and 14, followed by aerosolized OVA challenge on days 28, 29, and 30.A total of 350 muscular larvae were orally infected on days −3, 25, and 31, whereas 40 000 newborn larvae were introduced via tail vein injection on day 15.Mice were euthanized and analyzed on day 37. B, Lung sections were prepared and stained with hematoxylin and eosin (H-E).Inflammatory cell infiltration and bronchiolar hyperplasia were semiquantitatively assessed on a scale from absent (0) to minimal (1), mild (2), moderate (3), marked (4), and massive (5).C, Lung sections were stained with periodic acid-Schiff (PAS), and mucus production and goblet cell hyperplasia were also evaluated.D, Total cells, macrophages, lymphocytes, eosinophils, and neutrophils in BALF were stained with DiffQuik and analyzed.Data are expressed as the mean ± SD from 5 independent experiments.***P < .001.B and C, One-way analysis of variance, followed by a Dunnett multiple-comparisons test.D, Two-way analysis of variance, followed by a Dunnett multiple-comparisons test.BALF, bronchoalveolar lavage fluid; OVA, ovalbumin.and OVA-immunoglobulin G1 (IgG1) levels were significantly decreased in the T spiralis infection group at day −3 but not in the NBL infection group at day 15.Furthermore, the levels of OVA-IgG and OVA-IgG1 were significantly reduced in the T spiralis infection group at day 25.Moreover, T spiralis infection reduced the level of OVA-immunoglobulin E (IgE), particularly in the T spiralis infection groups at days −3 and 25.

Anti-inflammatory Cytokines Induced by T spiralis Adults Suppress Type 2 Inflammation
To further evaluate the immunomodulatory ability of T spiralis adults, we examined anti-and proinflammatory cytokine levels.In lung tissues, T spiralis significantly reduced the mRNA expression of thymic stromal lymphopoietin (TSLP) and interleukins 5, 25, and 33 (IL-5, IL-25, and IL-33; Figure 3B).In particular, T spiralis infection on day 31 had mildly affected the expression of IL-5, TSLP, and IL-33 as compared with the other treatments.OVA significantly promoted the levels of Th2 cytokines (interleukin 4 [IL-4], IL-5, and interleukin 13 [IL-13]) in BALF (Figure 3C).Furthermore, T spiralis infection lowered IL-4, IL-5, and IL-13 levels in BALF, with the T spiralis infection group at day −3 exhibiting lower cytokine levels than the T spiralis infection group at day 15.According to cytokine changes in the BALF, the T spiralis infection groups showed decreased levels of IL-4, IL-5, and IL-13 in serum, whereas interferon γ levels were not significantly altered.In contrast, the T spiralis infection upregulated IL-10 and TGF-β1 levels.Generally, the T spiralis infection group at day −3 exhibited the highest level of TGF-β1, and the T spiralis infection group at day 25 had the highest level of IL-10 among the 4 groups.

T spiralis Adults Affect Lung Immune Cells in OVA-Induced Allergic Models
We conducted flow cytometry analysis to assess changes in eosinophils, neutrophils, CD103 + dendritic cells (DCs), CD11b + DCs, alveolar macrophages (AMs), and interstitial macrophages (IMs) in the mouse lung (Supplementary Figure 3A and 3B).The OVA group exhibited higher proportions of eosinophils (CD11c − , CD11b + , and SiglecF + ) and neutrophils (CD11c − , CD11b + , and Ly6G + ) as compared with the control groups (Figure 4A and 4D).The proportion of eosinophils decreased in the T spiralis infection groups at days −3, 15, and 25 but did not change significantly in the day 31 group vs the OVA group.However, the proportion of neutrophils increased only in the T spiralis infection group on day 15 when compared with the OVA group (Figure 4B and 4D).Previous studies have categorized lung macrophages into 2 subsets: AMs (MHCII + , CD11c hi , CD11b int , and CD24 − ) and IMs (MHCII + , CD11b hi , and CD24 − ) [16].Sensitization with alum or OVA significantly reduced the proportion of AMs while increasing the proportion of IMs (Figure 4C and 4D).The proportions of IMs were significantly lower in the T spiralis infection groups at days −3, 15, and 25 vs the OVA group.Yet, only the group infected with T spiralis at day 25 exhibited a higher proportion of AMs than the OVA group.In lung tissues, CD103 + DCs (MHCII + , CD11c hi , CD11b + , and CD24 + ) and CD11b + DCs (MHCII + , CD11b hi , and CD24 + ) are the primary antigen-presenting cells that induce T-cell activation.The results demonstrated that OVA sensitization decreased the proportion of CD103 + DCs while increasing the proportion of CD11b + DCs.Only T spiralis infection on day −3 promoted the proportion of CD103 + DCs as compared with the OVA group.In contrast, T spiralis infection significantly reduced the proportion of CD11b + DCs.
To further investigate the involvement of T cells and macrophages in lung tissues, we employed anti-CD4 and anti-F4/80 antibodies for cell labeling.T spiralis infection mitigated cell infiltration and decreased the number of T cells, particularly in the infection groups on days −3 and 25 as compared with the OVA group.However, no significant change in the number of macrophages was observed (Figure 5).

T spiralis Adults Affect Lymphocytes of the Spleen and Peritoneal Macrophages
To evaluate the effect of lymphocytes and macrophages, we detected the differentiation of splenocyte subsets and peritoneal macrophages (Supplementary Figure 1B and 1C).T spiralis infection on days −3 and 25 significantly reduced the proportion of CD45 + cells in the spleen (Figure 6A and 6D).Examination of the changes in B-and T-cell subsets in OVA-induced allergic mice postinfection showed a higher number of B cells (CD45 + , B220 + , and CD4 − ) in the OVA group than the saline group.However, infection with T spiralis on days −3 and 25 significantly decreased the proportion of B cells (Figure 6B and 6D).Conversely, T spiralis infection increased the proportion of CD4 + T cells (CD45 + and CD4 + ), particularly on days −3 and 25.The proportion of Treg cells (CD45 + , CD4 + , and FoxP3 + ) in the T spiralis infection groups was significantly higher than that in the OVA group, particularly on days −3 and 25 (Figure 6C and 6D).Furthermore, peritoneal macrophages are reported to induce an immunosuppressive response during the intestinal phase of T spiralis infection [17].Significantly fewer F4/80 + macrophages were observed in the OVA group than the saline group, indicating that alum/OVA sensitization affected the proliferation of peritoneal macrophages (Figure 7A and 7C).Although T spiralis infection increased the proportion of macrophages, this change was not statistically significant in the NBL infection group at day 15.Similarly, T spiralis infection induced a higher proportion of CD16/32 + and CD206 + macrophages than the OVA control group (Figure 7B and 7C).Overall, these findings illustrate that T spiralis effectively modulated the host's immune response to suppress inflammation.

DISCUSSION
This study revealed the capacity of early-stage ESPs to induce alternative activation of macrophages, characterized by an upregulated CD206 expression and the production of antiinflammatory cytokines, such as IL-10 and TGF-β1.Among the time points studied, ESPs from AD3 exhibited the strongest induction capacity, possibly related to their role in helping NBL evade the host's immune response.Furthermore, ESPs, being complex heterologous antigens, triggered an immune response [18], including an increase in proinflammatory cytokine levels such as IL-12p70 and IL-1β.Therefore, we focused on understanding the immunosuppressive capacity of early T spiralis infection stages in the context of allergic diseases.
We examined the immunomodulatory effects of the migration and muscular stages during the challenge stage by injecting NBL into the tail vein.Using NBL infection as a control, we evaluated the immunoregulatory effect of intestinal adults on allergic inflammation during the sensitization stage.Our findings confirmed that adults alleviated the infiltration of inflammatory cells, reduced mucus production, and inhibited bronchiolar wall proliferation.Additionally, adults significantly lowered the levels of OVA-specific antibodies (IgG, IgG1, and IgE) during the sensitization and challenge stages.B cells were pivotal in antibody class switching, as influenced by various cytokines, including IL-4, which regulates class switching to IgE [19][20][21].The reduction in OVA-specific IgE levels was associated with the suppression of the type 2 immune response and a decrease in IL-4 levels.Interestingly, significant changes in OVA-specific IgG and IgG1 levels were observed in the groups infected on days −3 and 25 vs the OVA group, paralleling changes in B-cell populations in the spleen.This suggests that adults possess the ability to regulate humoral immunity.This study demonstrated that T spiralis reduces the mRNA expression levels of alarm cytokines (IL-25, IL-33, and TSLP), which are crucial in inducing the type 2 innate immune response.Furthermore, it resulted in decreased levels of Th2 cytokines (IL-4, IL-5, and IL-13) after infection, while interferon γ levels remained largely unchanged.In contrast, T spiralis induced high levels of anti-inflammatory cytokines (IL-10 and TGF-β1).Therefore, we concluded that the inhibitory effect of T spiralis on type 2 inflammation primarily relies on immunosuppression rather than a Th1-type immune response.The host's immune environment during the 3 allergy stages exhibited variations, and these different survival conditions faced by T spiralis in the 4 groups led to diverse immunomodulatory capacities of the worms.To comprehend the immunoregulatory effects on lung tissues, it was imperative to investigate changes in various immune cells within the lung.Although it is debated whether neutrophils contribute to the type 2 immune response, the elevated levels of interleukins 6, 8, and 17 in the lungs drove neutrophils into the tissue, potentially amplifying inflammation [22,23].In our study, the increased presence of neutrophils in the lungs induced by alum/OVA is likely associated with the development of inflammation.Interestingly, NBL infection before the challenge stage led to an increased influx of neutrophils into the lungs, possibly linked to the circulation of NBL through lung tissue.Additionally, eosinophils are crucial in allergic inflammation [24].Cytokines such as IL-5 and granulocyte-macrophage colony-stimulating factor are critical in eosinophil differentiation and maturation.Activated eosinophils present processed antigens to T cells, stimulating cell proliferation, and release various proinflammatory substances that amplify the inflammatory response, such as IL-4, IL-5, and IL-13 and major basic proteins 1 and 2 [25,26].This study demonstrated that T spiralis infection prior to the challenge stage reduced the recruitment of eosinophils, which is important in limiting inflammation and promoting tissue homeostasis.Furthermore, T spiralis reduced the number of infiltrating CD4 + T cells around the bronchioles.
Lung macrophages can be categorized into 2 populations-AMs and IMs.AMs located in the alveolar space are exposed to the external environment and serve to eliminate potentially harmful substances [27].These AMs are derived from embryonic progenitors and have the capacity for self-proliferation under lung homeostasis [28].During the challenge phase of allergy, AMs upregulate YM1 and Arg1 expression, inducing an immunosuppressive response to limit the lung DC function and antigen-specific antibody production [29][30][31].We found a significant decrease in the AM proportion in the OVA group as compared with the saline group.The AM proportion was promoted by adults only before the challenge phase.Additionally, IMs within the lung interstitium are maintained by circulating monocytes and are considered to prevent immune responses against harmless inhaled antigens [32].There is considerable debate regarding which subsets of IMs promote or suppress inflammation [30,33,34].Our findings revealed that OVA induced an increase in the proportion of IMs in mouse lungs, contrasting with the trend observed for AMs.Adults significantly reduced the IM population during the allergy development, while NBL had no obvious effect, indicating that the inhibition of IM recruitment depended on the immunoregulation of adults.
Th2 cells are pivotal in aggravating the inflammatory response through the production of cytokines such as IL-4, IL-5, and IL-13 [35].DCs are crucial for initiating the adaptive immune response because of their robust migration and antigen presentation capabilities [36].In the lungs, DCs are typically divided into 3 subsets at a steady state: plasmacytoid DCs and conventional DCs (comprising CD11b + cDCs and CD103 + DCs).Under inflammatory conditions, monocyte-derived DCs are recruited into lung tissues and produce proinflammatory chemokines to attract eosinophils and effector T cells in the airways.Monocyte-derived DCs are similar to CD11b + cDCs, which participate in and exacerbate allergic inflammation [37,38].Consequently, we investigated these 2 subsets as CD11b + DCs.CD11b + DCs are reported to prime the type 2 adaptive immune response, whereas CD103 + DCs promote the type 1 adaptive immune response, helping to suppress excessive type 2 inflammation through the release of IL-12 [39].In our study, the number of CD11b + DCs increased in the lungs following OVA stimulation, while the proportion of CD103 + DCs decreased.T spiralis infection significantly reduced the proportion of CD11b + DCs, which was associated with a subsequent weakening of the adaptive type 2 immune response.Interestingly, the proportion of CD103 + DCs increased significantly when T spiralis infection occurred before the sensitization stage.The immunosuppressive environment induced by adult worms reduced proinflammatory cell subsets in the lung.Additionally, adults demonstrated the ability to maintain immune homeostasis by promoting the differentiation of AMs and CD103 + DCs in the lungs.However, this immunomodulatory capacity was effective only before OVA challenge.
Helminths create an immunosuppressive microenvironment to survive within parasitic tissues.Additionally, helminths can suppress inflammation in other tissues, primarily relying on anti-inflammatory cytokines [5,40].Anti-inflammatory cytokines are primarily produced by Treg cells and alternatively activated macrophages [5,17,41,42].CD4 + Foxp3 + Treg cells induced by T spiralis possess a stronger ability to migrate and suppress the immune response than normal Treg cells [43].According to flow cytometry analysis, adult worms significantly reduced the CD45 + immune cell population in the spleen before the onset of allergic inflammation.In contrast, T spiralis infection after the challenge phase had no effect on CD45 + immune cells, and NBL infection increased the proportion of CD45 + immune cells.Therefore, we concluded that immunoregulation by intestinal adults is crucial in suppressing lung inflammation before allergic inflammation is activated.Consistent with the changes observed in OVA-specific IgG and IgG1, adults reduced the proportion of B220 + B cells before allergic inflammation was activated.Conversely, T spiralis infection increased the proportion of Treg cells.In the T spiralis infection groups on days −3 and 25, 70% of CD4 + T cells were Treg cells, indicating a significant reduction in T helper cells.Thus, Treg cells induced by adults are vital in suppressing inflammation.Furthermore, during the intestinal phase of T spiralis infection, the immune microenvironment in the peritoneal cavity is significant in anti-inflammation [44,45].Macrophages constitute a significant proportion of the peritoneal cavity's immune cells, with alternatively activated macrophages being primarily responsible for inducing an anti-inflammatory response [17].Our results demonstrated that the use of alum/OVA disrupted immune homeostasis in the peritoneal cavity, leading to a decreased proportion of macrophages.Adults had the ability to restore the proportion of macrophages, whereas NBL infection did not.Additionally, T spiralis infection upregulated CD206 expression, suggesting an increase in the proportion of alternatively activated macrophages.
In conclusion, T spiralis adults induce an immunosuppressive response through the differentiation of alternatively activated macrophages and Treg cells, as well as by increasing the levels of IL-10 and TGF-β1.This immunosuppression effectively suppresses excessive type 2 immune responses.Moreover, before the onset of allergic inflammation, adults contribute to maintaining the balance of immune cells in the lung and protecting pulmonary immune homeostasis.To conclude, this study elucidated the immunoregulatory effects of T spiralis adults in inhibiting the development of allergic diseases.It provides insights into how the immunoregulatory capabilities of these worms can be harnessed for potential treatments of allergic diseases, such as through infection with single-sex T spiralis or the utilization of adult-derived proteins.

Figure 2 .
Figure 2.Trichinella spiralis mitigates allergic inflammation in the lungs.A, Experimental design.Mice underwent sensitization with OVA/alum via intraperitoneal injection on days 0, 7, and 14, followed by aerosolized OVA challenge on days 28, 29, and 30.A total of 350 muscular larvae were orally infected on days −3, 25, and 31, whereas 40 000 newborn larvae were introduced via tail vein injection on day 15.Mice were euthanized and analyzed on day 37. B, Lung sections were prepared and stained with hematoxylin and eosin (H-E).Inflammatory cell infiltration and bronchiolar hyperplasia were semiquantitatively assessed on a scale from absent (0) to minimal (1), mild (2), moderate (3), marked (4), and massive(5).C, Lung sections were stained with periodic acid-Schiff (PAS), and mucus production and goblet cell hyperplasia were also evaluated.D, Total cells, macrophages, lymphocytes, eosinophils, and neutrophils in BALF were stained with DiffQuik and analyzed.Data are expressed as the mean ± SD from 5 independent experiments.***P < .001.B and C, One-way analysis of variance, followed by a Dunnett multiple-comparisons test.D, Two-way analysis of variance, followed by a Dunnett multiple-comparisons test.BALF, bronchoalveolar lavage fluid; OVA, ovalbumin.

Figure 5 .
Figure 5. Immunofluorescence of CD4 + T cells and macrophages in lung.A, Immunostained lung sections show F4/80, CD4, and Hoechst.CD4 + T cells and F4/80 + macrophages are indicated by arrows.B, The number of CD4 + T cells per square millimeter was counted.Data are expressed as the mean ± SD from 5 independent experiments.***P < .001.One-way analysis of variance, followed by a Dunnett multiple-comparisons test.OVA, ovalbumin.

Figure 6 .
Figure 6.Suppression of immune cell proliferation and promotion of Treg cell differentiation by Trichinella spiralis adults in the spleen.Flow cytometry analysis of (A) CD45 + immune cells, (B) B220 + B cells and CD4 + T cells, and (C ) Foxp3 + Treg cells in the spleen.D, Proportion of CD45 + immune cells in single cells, B cells and CD4 + T cells in CD45 + cells, and Treg cells in CD4 + T cells.Data are expressed as the mean ± SD from 5 independent experiments.ns, not significant.*P < .05. **P < .01. ***P < .001.One-way analysis of variance, followed by a Dunnett multiple-comparisons test.OVA, ovalbumin; SSC-A, side scatter area.