Escherichia coli enterotoxin modulates immunity but fails to prevent autoimmune hemolytic anemia in NZB mice

Escherichia coli enterotoxin modulates immunity but fails to prevent autoimmune hemolytic anemia in NZB mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Escherichia coli enterotoxin modulates immunity but fails to prevent autoimmune hemolytic anemia in NZB mice Abdel-Rahman Youssef This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7656673/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 18 You are reading this latest preprint version Abstract Background The B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a strong immunological modulator that enhances T helper-1 (Th1)-mediated autoimmune diseases. This study aimed to evaluate the effectiveness of inhaled EtxB in treating autoimmune hemolytic anemia (AIHA) in New Zealand Black (NZB) mice, which naturally develop AIHA. Methods Eight NZB mice received 20 µg of recombinant EtxB intranasally for four consecutive days, while the control group inhaled phosphate-buffered saline (PBS). The levels of total IgG, IgG1, and IgG2a antibodies bound to red blood cells (RBC) were measured by cellular ELISA at 6 and 12 weeks. Hematocrit values were assessed at 12 weeks and 4 months using a Hawksley Micro-hematocrit reader. Results After 6 weeks, EtxB-treated mice exhibited higher levels of RBC-bound IgG and IgG2a, along with a significantly lower IgG1/IgG2a ratio ( P 0.05), indicating a temporary immune modulation. EtxB did not delay the onset of anemia, as hematocrit readings showed no significant differences between groups at any time point ( P > 0.05). Conclusion This study demonstrated that inhaling EtxB temporarily modulated the immune response at 6 weeks but did not sustain this effect beyond 12 weeks or prevent anemia. EtxB Autoimmune hemolytic anemia NZB mice Figures Figure 1 Figure 2 Figure 3 1. Introduction Activated CD4 T cells produce T helper-1 (Th1) and T helper-2 (Th2) cytokines that regulate immune responses. Th1 cytokines include interferon-gamma (IFN-γ), IL-2, and tumor necrosis factor-alpha (TNF-α), whereas Th2 cytokines include IL-4, IL-5, IL-10, and IL-13. Generally, Th1 cytokines activate cell-mediated immunity, while Th2 cytokines stimulate humoral immunity (Abbas et al., 1996 ; O’Garra, 1998 ). In mice, the production of IgG2a antibodies is induced by Th1-dependent IFN-γ. At the same time, B cells are regulated by IL-4 for the secretion of IgG1 antibodies, rendering IgG2a and IgG1 immunoglobulin isotypes as indicators for Th1 and Th2 responses, respectively (Germannet al., 1993 ; Stevens et al., 1988 ). In New Zealand Black (NZB) mice, autoimmune hemolytic anemia (AIHA) occurs spontaneously. In murine AIHA, red blood cells (RBC) are lysed due to the production of autoantibodies against them. High levels of IL-2, IFN-γ, and TNF-α indicate a Th1 cytokine profile, which predominates during the development of AIHA in NZB mice (Hallet al., 2012 ). The balance between Th1 and Th2 responses is critical. The dominance of Th1 responses may enhance autoantibody production and lead to hemolytic anemia, while a shift toward Th2 responses might alleviate these effects (J. Chen et al., 2012 ). The administration of specific peptides derived from autoantigens has shown promise in ameliorating anemia by shifting the immune response towards a more protective Th2 profile (Shen et al., 2003 ). Furthermore, NZB mice treated with pIL-4 delayed the development of anemia compared with plasmid-treated controls (Youssef et al., 2005 ). The B subunit of Escherichia coli heat-labile enterotoxin (EtxB) has dual roles as an immunomodulator and a toxin. EtxB attaches to GM1-ganglioside receptors on host cells, just like the cholera toxin B component (CtxB), which facilitates the entry of the poisonous A subunit into cells. The nontoxic EtxB and CtxB have important immunomodulatory properties that can reduce inflammatory reactions associated with autoimmune diseases such as diabetes and rheumatoid arthritis (Aman et al., 2001 ; Lesieur et al., 2002 ). EtxB is a strong immunomodulatory agent that suppresses Th1-associated inflammatory responses by inducing immunological deviation pathways (Salmond et al., 2004 ). It has been demonstrated that EtxB is a strong adjuvant that can activate T cells. This activation results from the induction of dendritic cells (DC), which prolongs the presentation of antigen to T cells and increases T cell activation (Ji et al., 2015a ). Furthermore, EtxB can modulate T cell responses by biasing the immune response to the Th2 profile (Conti et al., 2023 ) and increasing regulatory T (Treg) cells (Bignon et al., 2017 ). These immunomodulatory effects raise the possibility of using EtxB to treat autoimmune and inflammatory diseases (Lesieur et al., 2002 ). Taking these findings together with those connecting EtxB to protection against other autoimmune disorders, the possibility that EtxB administration could affect the development of AIHA in NZB mice was investigated in this study. 2. Materials and Methods 2.1. Mice Six-week-old NZB (H-2d) mice were obtained from Harlan Laboratories (Bicester, UK) and used in this study. The mice were housed in the animal facilities of Bristol University and maintained in a pathogen-free environment. The "Principles of Laboratory Animal Care" were consistently adhered to, and all animal experiments were conducted in accordance with UK Home Office regulations. 2.2. Administration of EtxB Recombinant EtxB was generously donated by Professor Neil Williams of the University of Bristol. Recombinant EtxB was purified from Vibrio sp. 60 (pMMB68), as previously reported (Amin & Hirst, 1994 ). A group of eight NZB mice received 20 µg of recombinant EtxB in phosphate-buffered saline (PBS) intranasally daily for four consecutive days, as mentioned previously (Donaldson et al., 2013 ). Six mice of the same age and sex were treated with PBS (Sigma, UK) and served as the control group. 2.3. Measurement of IgG autoantibodies Bound to RBC The total IgG, IgG1, and IgG2a antibodies bound to the surface of RBC were quantified using cellular ELISA, which is based on the direct enzyme-linked antiglobulin test (DELAT) as previously reported (Youssef et al., 2005 ). Briefly, RBC were washed in PBS that had been preheated to 37°C. A 10% suspension of RBC was fixed with 0.5% glutaraldehyde (Sigma, UK). Sheep anti-mouse IgG, IgG1, or IgG2a antibodies (The Binding Site, UK) were added to 50 µl aliquots of a 2% suspension of fixed RBC in the wells of a round-bottomed microtiter plate (Corning, USA) and incubated at 37°C for 1 hour. After washing, the RBCs were treated with an alkaline-phosphatase-conjugated donkey anti-sheep antibody (Sigma, UK) for 1 hour at 37°C. Following washing, the plate was incubated with p-Nitrophenyl Phosphate (pNPP) substrate (Sigma, UK) for an hour at 37°C. The supernatant was transferred into a flat-bottom microtiter plate (Corning, USA), and the absorbance was measured at 405 nm using a microplate reader (Bio-Rad Laboratories Inc., USA). 2.4. Hematocrit Values A 70 µl of blood was obtained from the lateral saphenous vein and collected in glass micro-hematocrit capillary sodium-heparinized tubes (Hawksley, UK). After spinning the tubes, a Hawksley Micro-hematocrit reader was used to determine the hematocrit value, which was expressed as the percentage of the RBC column's relative height. 2.5. Statistical Analysis Microsoft Excel and GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA) were used for data collection, statistical analysis, and graphing. The data, presented as mean and standard deviation (SD), were analyzed using an unpaired t-test and one-way analysis of variance (ANOVA). A P -value of less than 0.05 was considered significant. 3. Results RBC-bound autoantibodies and hematocrit values were assessed in NZB mice after receiving EtxB intranasally compared to the control mice that received PBS. The mean and standard deviation with the P -value for the comparison of RBC-bound immunoglobulins in mice that inhaled EtxB or PBS after 6 and 12 weeks are shown in Table 1 . After 6 weeks of EtxB inhalation (Fig. 1 A), EtxB-treated mice had significantly higher levels of RBC-bound total IgG, IgG1, and IgG2a antibodies than PBS-treated controls ( P 0.05) after 12 weeks (Fig. 1 B). After 6 weeks of EtxB inhalation, the mice had an IgG1/IgG2a ratio of 0.31, compared to 0.36 in the PBS control group (Fig. 2 A). This difference was statistically significant ( P = 0.02), but the ratio in both groups was < 0.5, suggesting a Th1-skewed immune response. By 12 weeks (Fig. 2 B), the average IgG1:IgG2a ratio was 0.85 in mice inhaled EtxB and 0.56 in the control group, indicating combined Th1 and Th2 responses, and this difference was not statistically significant ( P > 0.05). Estimates of the hematocrit values after 12 weeks and 4 months after EtxB inhalation (Fig. 3 ) showed that EtxB did not delay the development of anemia since hematocrit values were not significantly different from those for control mice ( P > 0·05). Table 1 Mean and standard deviation (SD) with the P -value for the comparison of RBC-bound immunoglobulins in mice inhaled B subunit of Escherichia coli heat-labile enterotoxin (EtxB) or phosphate-buffered saline (PBS) after 6 and 12 weeks. Week 6 Mean ± SD P-value Week 12 Mean ± SD P -value Control EtxB Control EtxB IgG 0.32 ± 0.03 0.39 ± 0.06 0.0354 IgG 0.80 ± 0.34 0.87 ± 0.23 0.6582 IgG1 0.13 ± 0.01 0.14 ± 0.01 0.0445 IgG1 0.21 ± 0.06 0.35 ± 0.19 0.1002 IgG2a 0.36 ± 0.03 0.46 ± 0.04 0.0002 IgG2a 0.38 ± 0.09 0.43 ± 0.06 0.2088 4. Discussion This study evaluated whether EtxB administration could be an effective therapy for preventing autoimmune hemolytic anemia in NZB mice. The results of the present study demonstrated that EtxB inhalation increased the levels of RBC-bound total IgG, Th1-dependent IgG2a, and Th2-dependent IgG1 and reduced the IgG1/IgG2a ratio at 6 weeks. However, this effect was not sustained after 12 weeks. In NZB mice, RBC autoantibodies against IgG2a are more pathogenic than those against IgG1 (Fossati-Jimack et al., 2000 ). The bias of an immune response toward T helper cells can be evaluated using the ratio of antigen-specific IgG1 to IgG2a antibodies, as well as cytokine profiling. A Th1-biased response is indicated by an IgG1:IgG2a ratio of less than 0.5, while a Th2-biased response is indicated by a ratio greater than 2. A mixed response is shown by ratios between 0.5 and 2 (Feltquate et al., 1997a ). In this study, after 6 weeks, the IgG1/IgG2a ratio in both the EtxB and PBS control groups was less than 0.5, suggesting a Th1-skewed immune response, whereas the ratio at 12 weeks was between 0.5 and 2, indicating a mixed Th1 and Th2 response. The shift in RBC autoantibody isotype from the pathogenic IgG2a type to IgG1 was assumed to be the cause of the EtxB treatment's therapeutic benefit (Fossati-Jimack et al., 2000 ), as NZB mice have a Th1-dominant cytokine profile throughout the course of AIHA (Hall et al., 2012 ). However, the findings of the current study challenge this idea, as no such change was observed. The increased RBC-bound autoantibodies after EtxB administration stem from enhanced B-cell activation. EtxB interaction with Ganglioside M1 (GM1) on B cells and DC notably promotes the proliferation of EtxB-specific CD4 T helper cells and cytokine production (Nashar et al., 2001 ). Additionally, when EtxB binds to B cells, there is an upregulation of CD25 and major histocompatibility complex (MHC) class II on B cells (Bone et al., 2002 ). Besides class II MHC expression, the costimulatory signals, such as CD86 and CD40, are also upregulated, which are essential for effective T cell activation and subsequent antibody production (Salmond et al., 2004 ; Williams et al., 1997 ). The current study showed that EtxB treatment did not prevent anemia in NZB mice. Conversely, EtxB treatment altered the immune responses and decreased clinical disease in several animal models of autoimmune disease (Luross et al., 2002 ; Ola & Williams, 2006 ; Raveney et al., 2008 ; Williams et al., 1997 ). Previous studies have demonstrated that EtxB influences immunological responses by skewing the immune response towards a Th2 profile (Conti et al., 2023 ) and enhancing Treg populations (Bignon et al., 2017 ). It has been demonstrated that EtxB preimmunization protects mice from murine experimental autoimmune uveoretinitis (EAU) induction by inhibiting Th1 responses (Raveney et al., 2008 ). Furthermore, EtxB increased IL-4 production, which induces differentiation of naive helper T cells to Th2 cells and activates B cells (Conti et al., 2023 ). Other studies have shown that EtxB-induced FOXP3 + Treg cell development which is essential in maintaining immune tolerance and preventing autoimmune diseases. It has previously been demonstrated that EtxB treatment results in increased Foxp3 + CD4 + Treg cells (Bignon et al., 2017 ; Donaldson et al., 2011 ) and alters innate cells by lowering the number of myeloid and DC precursors in secondary lymphoid organs (Bignon et al., 2017 ; Ji et al., 2015b ). Moreover, EtxB increased the expression of IL-10 and transforming growth factor-β 1 (TGF-β 1 ) in CD11b + cells, which increased in Foxp3 + T cells (Bignon et al., 2017 ; W. J. Chen et al., 2003 ; Donaldson et al., 2011 ). In the context of allergic disorders, EtxB has been demonstrated to prevent the development of allergic airway disease by boosting CD4 + Treg cell counts and allergen-specific Treg responses (Donaldson et al., 2013 ; Zhang et al., 2014 ). Although EtxB may modulate immune responses in several animal models, this does not always translate into a successful treatment course for autoimmune diseases. In this study, EtxB inhalation modulated the immune response in NZB mice after 6 weeks, but it did not stop the development of anemia. This supports the findings of Raveney et al., who demonstrated that EtxB restricted the quantity and activation state of retinal-invasive immune cells before EAU induction. However, the course of the disease was unaffected by EtxB treatment following EAU induction (Raveney et al., 2008 ). This could be because the components of the immune system function differently in the presence of autoimmune disease. Although animal models can replicate human diseases and pathologies, they have certain limitations, such as species differences and limited genetic diversity. Additionally, not all biological processes can be directly applied to humans because human physiology is more complex than that of animals. 5. Conclusion EtxB inhalation modulated the immune response in NZB mice at 6 weeks, but this effect was not sustained at 12 weeks and did not prevent autoimmune hemolytic anemia in NZB mice. Declarations Conflicts of Interest: No financial or non-financial interests exist. Ethical approval: This study was approved by the experimental animals’ administration at the University of Bristol, UK. Statement of animal rights The "Principles of Laboratory Animal Care" were always adhered to, and all animal experiments were carried out under UK Home Office rules. Statement of informed consent There are no human subjects in this study, and informed consent is not applicable. 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11:21:19","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":88471,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7656673/v1/7755ea5084881dd23adf57e6.html"},{"id":93929722,"identity":"64ab25e0-3a9d-4992-b643-5f21ba092c5c","added_by":"auto","created_at":"2025-10-20 11:29:19","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":98403,"visible":true,"origin":"","legend":"\u003cp\u003eRBC-bound immunoglobulins in mice inhaled EtxB or PBS. At six weeks (A), EtxB-treated mice had higher levels of total IgG, IgG1, and IgG2a antibodies than PBS-treated controls (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). At 12 weeks (B), the change was insignificant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7656673/v1/dd79fc363acab56a2b5ac1e5.jpeg"},{"id":93929723,"identity":"d83746cb-3b05-47ad-828e-f0054a3af329","added_by":"auto","created_at":"2025-10-20 11:29:19","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":86800,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of RBC-bound IgG1 to IgG2a ratio in mice inhaled with PBS control. At 6 weeks, (A) the ratio was reduced significantly (\u003cem\u003eP\u003c/em\u003e = 0·02). By 12 weeks (B), no significant difference was observed between the EtxB and control groups (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0·05).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7656673/v1/b016fbf7fc95de0444fa1088.jpeg"},{"id":93929031,"identity":"6e944884-a083-47e4-9396-982ef988deb0","added_by":"auto","created_at":"2025-10-20 11:21:19","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60406,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the hematocrit values of individual NZB mice after they inhaled EtxB or PBS. The hematocrit was measured after 12 weeks (A) and 4 months (B). No significant difference between the control and treated groups (\u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7656673/v1/39251d81ab77489e15fffaa2.jpeg"},{"id":93929725,"identity":"92c74495-34dc-4f17-bcc8-292b7082cf40","added_by":"auto","created_at":"2025-10-20 11:29:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":811150,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7656673/v1/c1f53d5d-0783-45ad-87ca-e7f326a86935.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Escherichia coli enterotoxin modulates immunity but fails to prevent autoimmune hemolytic anemia in NZB mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eActivated CD4 T cells produce T helper-1 (Th1) and T helper-2 (Th2) cytokines that regulate immune responses. Th1 cytokines include interferon-gamma (IFN-γ), IL-2, and tumor necrosis factor-alpha (TNF-α), whereas Th2 cytokines include IL-4, IL-5, IL-10, and IL-13. Generally, Th1 cytokines activate cell-mediated immunity, while Th2 cytokines stimulate humoral immunity (Abbas et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; O\u0026rsquo;Garra, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In mice, the production of IgG2a antibodies is induced by Th1-dependent IFN-γ. At the same time, B cells are regulated by IL-4 for the secretion of IgG1 antibodies, rendering IgG2a and IgG1 immunoglobulin isotypes as indicators for Th1 and Th2 responses, respectively (Germannet al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Stevens et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1988\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn New Zealand Black (NZB) mice, autoimmune hemolytic anemia (AIHA) occurs spontaneously. In murine AIHA, red blood cells (RBC) are lysed due to the production of autoantibodies against them. High levels of IL-2, IFN-γ, and TNF-α indicate a Th1 cytokine profile, which predominates during the development of AIHA in NZB mice (Hallet al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The balance between Th1 and Th2 responses is critical. The dominance of Th1 responses may enhance autoantibody production and lead to hemolytic anemia, while a shift toward Th2 responses might alleviate these effects (J. Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The administration of specific peptides derived from autoantigens has shown promise in ameliorating anemia by shifting the immune response towards a more protective Th2 profile (Shen et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Furthermore, NZB mice treated with pIL-4 delayed the development of anemia compared with plasmid-treated controls (Youssef et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe B subunit of \u003cem\u003eEscherichia coli heat-labile\u003c/em\u003e enterotoxin (EtxB) has dual roles as an immunomodulator and a toxin. EtxB attaches to GM1-ganglioside receptors on host cells, just like the cholera toxin B component (CtxB), which facilitates the entry of the poisonous A subunit into cells. The nontoxic EtxB and CtxB have important immunomodulatory properties that can reduce inflammatory reactions associated with autoimmune diseases such as diabetes and rheumatoid arthritis (Aman et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Lesieur et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEtxB is a strong immunomodulatory agent that suppresses Th1-associated inflammatory responses by inducing immunological deviation pathways (Salmond et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). It has been demonstrated that EtxB is a strong adjuvant that can activate T cells. This activation results from the induction of dendritic cells (DC), which prolongs the presentation of antigen to T cells and increases T cell activation (Ji et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e). Furthermore, EtxB can modulate T cell responses by biasing the immune response to the Th2 profile (Conti et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and increasing regulatory T (Treg) cells (Bignon et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These immunomodulatory effects raise the possibility of using EtxB to treat autoimmune and inflammatory diseases (Lesieur et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Taking these findings together with those connecting EtxB to protection against other autoimmune disorders, the possibility that EtxB administration could affect the development of AIHA in NZB mice was investigated in this study.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Mice\u003c/h2\u003e\u003cp\u003eSix-week-old NZB (H-2d) mice were obtained from Harlan Laboratories (Bicester, UK) and used in this study. The mice were housed in the animal facilities of Bristol University and maintained in a pathogen-free environment. The \"Principles of Laboratory Animal Care\" were consistently adhered to, and all animal experiments were conducted in accordance with UK Home Office regulations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Administration of EtxB\u003c/h2\u003e\u003cp\u003eRecombinant EtxB was generously donated by Professor Neil Williams of the University of Bristol. Recombinant EtxB was purified from \u003cem\u003eVibrio\u003c/em\u003e sp. 60 (pMMB68), as previously reported (Amin \u0026amp; Hirst, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). A group of eight NZB mice received 20 \u0026micro;g of recombinant EtxB in phosphate-buffered saline (PBS) intranasally daily for four consecutive days, as mentioned previously (Donaldson et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Six mice of the same age and sex were treated with PBS (Sigma, UK) and served as the control group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Measurement of IgG autoantibodies Bound to RBC\u003c/h2\u003e\u003cp\u003eThe total IgG, IgG1, and IgG2a antibodies bound to the surface of RBC were quantified using cellular ELISA, which is based on the direct enzyme-linked antiglobulin test (DELAT) as previously reported (Youssef et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Briefly, RBC were washed in PBS that had been preheated to 37\u0026deg;C. A 10% suspension of RBC was fixed with 0.5% glutaraldehyde (Sigma, UK). Sheep anti-mouse IgG, IgG1, or IgG2a antibodies (The Binding Site, UK) were added to 50 \u0026micro;l aliquots of a 2% suspension of fixed RBC in the wells of a round-bottomed microtiter plate (Corning, USA) and incubated at 37\u0026deg;C for 1 hour. After washing, the RBCs were treated with an alkaline-phosphatase-conjugated donkey anti-sheep antibody (Sigma, UK) for 1 hour at 37\u0026deg;C. Following washing, the plate was incubated with p-Nitrophenyl Phosphate (pNPP) substrate (Sigma, UK) for an hour at 37\u0026deg;C. The supernatant was transferred into a flat-bottom microtiter plate (Corning, USA), and the absorbance was measured at 405 nm using a microplate reader (Bio-Rad Laboratories Inc., USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Hematocrit Values\u003c/h2\u003e\u003cp\u003eA 70 \u0026micro;l of blood was obtained from the lateral saphenous vein and collected in glass micro-hematocrit capillary sodium-heparinized tubes (Hawksley, UK). After spinning the tubes, a Hawksley Micro-hematocrit reader was used to determine the hematocrit value, which was expressed as the percentage of the RBC column's relative height.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Statistical Analysis\u003c/h2\u003e\u003cp\u003eMicrosoft Excel and GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA) were used for data collection, statistical analysis, and graphing. The data, presented as mean and standard deviation (SD), were analyzed using an unpaired t-test and one-way analysis of variance (ANOVA). A \u003cem\u003eP\u003c/em\u003e-value of less than 0.05 was considered significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eRBC-bound autoantibodies and hematocrit values were assessed in NZB mice after receiving EtxB intranasally compared to the control mice that received PBS. The mean and standard deviation with the \u003cem\u003eP\u003c/em\u003e-value for the comparison of RBC-bound immunoglobulins in mice that inhaled EtxB or PBS after 6 and 12 weeks are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. After 6 weeks of EtxB inhalation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), EtxB-treated mice had significantly higher levels of RBC-bound total IgG, IgG1, and IgG2a antibodies than PBS-treated controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, this difference was not maintained (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) after 12 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eAfter 6 weeks of EtxB inhalation, the mice had an IgG1/IgG2a ratio of 0.31, compared to 0.36 in the PBS control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This difference was statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02), but the ratio in both groups was \u0026lt;\u0026thinsp;0.5, suggesting a Th1-skewed immune response. By 12 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), the average IgG1:IgG2a ratio was 0.85 in mice inhaled EtxB and 0.56 in the control group, indicating combined Th1 and Th2 responses, and this difference was not statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eEstimates of the hematocrit values after 12 weeks and 4 months after EtxB inhalation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) showed that EtxB did not delay the development of anemia since hematocrit values were not significantly different from those for control mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0\u0026middot;05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean and standard deviation (SD) with the \u003cem\u003eP\u003c/em\u003e-value for the comparison of RBC-bound immunoglobulins in mice inhaled B subunit of \u003cem\u003eEscherichia coli\u003c/em\u003e heat-labile enterotoxin (EtxB) or phosphate-buffered saline (PBS) after 6 and 12 weeks.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eWeek 6\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eWeek 12\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEtxB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eEtxB\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIgG\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0354\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eIgG\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.6582\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIgG1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0445\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eIgG1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.1002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIgG2a\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eIgG2a\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.2088\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study evaluated whether EtxB administration could be an effective therapy for preventing autoimmune hemolytic anemia in NZB mice. The results of the present study demonstrated that EtxB inhalation increased the levels of RBC-bound total IgG, Th1-dependent IgG2a, and Th2-dependent IgG1 and reduced the IgG1/IgG2a ratio at 6 weeks. However, this effect was not sustained after 12 weeks.\u003c/p\u003e\u003cp\u003eIn NZB mice, RBC autoantibodies against IgG2a are more pathogenic than those against IgG1 (Fossati-Jimack et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The bias of an immune response toward T helper cells can be evaluated using the ratio of antigen-specific IgG1 to IgG2a antibodies, as well as cytokine profiling. A Th1-biased response is indicated by an IgG1:IgG2a ratio of less than 0.5, while a Th2-biased response is indicated by a ratio greater than 2. A mixed response is shown by ratios between 0.5 and 2 (Feltquate et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1997a\u003c/span\u003e). In this study, after 6 weeks, the IgG1/IgG2a ratio in both the EtxB and PBS control groups was less than 0.5, suggesting a Th1-skewed immune response, whereas the ratio at 12 weeks was between 0.5 and 2, indicating a mixed Th1 and Th2 response. The shift in RBC autoantibody isotype from the pathogenic IgG2a type to IgG1 was assumed to be the cause of the EtxB treatment's therapeutic benefit (Fossati-Jimack et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), as NZB mice have a Th1-dominant cytokine profile throughout the course of AIHA (Hall et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, the findings of the current study challenge this idea, as no such change was observed.\u003c/p\u003e\u003cp\u003eThe increased RBC-bound autoantibodies after EtxB administration stem from enhanced B-cell activation. EtxB interaction with Ganglioside M1 (GM1) on B cells and DC notably promotes the proliferation of EtxB-specific CD4 T helper cells and cytokine production (Nashar et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Additionally, when EtxB binds to B cells, there is an upregulation of CD25 and major histocompatibility complex (MHC) class II on B cells (Bone et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Besides class II MHC expression, the costimulatory signals, such as CD86 and CD40, are also upregulated, which are essential for effective T cell activation and subsequent antibody production (Salmond et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Williams et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe current study showed that EtxB treatment did not prevent anemia in NZB mice. Conversely, EtxB treatment altered the immune responses and decreased clinical disease in several animal models of autoimmune disease (Luross et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Ola \u0026amp; Williams, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Raveney et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Williams et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Previous studies have demonstrated that EtxB influences immunological responses by skewing the immune response towards a Th2 profile (Conti et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and enhancing Treg populations (Bignon et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It has been demonstrated that EtxB preimmunization protects mice from murine experimental autoimmune uveoretinitis (EAU) induction by inhibiting Th1 responses (Raveney et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Furthermore, EtxB increased IL-4 production, which induces differentiation of naive helper T cells to Th2 cells and activates B cells (Conti et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOther studies have shown that EtxB-induced FOXP3\u003csup\u003e+\u003c/sup\u003e Treg cell development which is essential in maintaining immune tolerance and preventing autoimmune diseases. It has previously been demonstrated that EtxB treatment results in increased Foxp3\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e Treg cells (Bignon et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Donaldson et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and alters innate cells by lowering the number of myeloid and DC precursors in secondary lymphoid organs (Bignon et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ji et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e). Moreover, EtxB increased the expression of IL-10 and transforming growth factor-β\u003csub\u003e1\u003c/sub\u003e (TGF-β\u003csub\u003e1\u003c/sub\u003e) in CD11b\u003csup\u003e+\u003c/sup\u003e cells, which increased in Foxp3\u003csup\u003e+\u003c/sup\u003e T cells (Bignon et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; W. J. Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Donaldson et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In the context of allergic disorders, EtxB has been demonstrated to prevent the development of allergic airway disease by boosting CD4\u0026thinsp;+\u0026thinsp;Treg cell counts and allergen-specific Treg responses (Donaldson et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although EtxB may modulate immune responses in several animal models, this does not always translate into a successful treatment course for autoimmune diseases. In this study, EtxB inhalation modulated the immune response in NZB mice after 6 weeks, but it did not stop the development of anemia. This supports the findings of Raveney et al., who demonstrated that EtxB restricted the quantity and activation state of retinal-invasive immune cells before EAU induction. However, the course of the disease was unaffected by EtxB treatment following EAU induction (Raveney et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This could be because the components of the immune system function differently in the presence of autoimmune disease.\u003c/p\u003e\u003cp\u003eAlthough animal models can replicate human diseases and pathologies, they have certain limitations, such as species differences and limited genetic diversity. Additionally, not all biological processes can be directly applied to humans because human physiology is more complex than that of animals.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eEtxB inhalation modulated the immune response in NZB mice at 6 weeks, but this effect was not sustained at 12 weeks and did not prevent autoimmune hemolytic anemia in NZB mice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of Interest:\u003c/h2\u003e\u003cp\u003eNo financial or non-financial interests exist.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e\u003cp\u003e This study was approved by the experimental animals\u0026rsquo; administration at the University of Bristol, UK.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatement of animal rights\u003c/strong\u003e\u003cp\u003e The \"Principles of Laboratory Animal Care\" were always adhered to, and all animal experiments were carried out under UK Home Office rules.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatement of informed consent\u003c/strong\u003e\u003cp\u003eThere are no human subjects in this study, and informed consent is not applicable.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYoussef AR performed the experiments, analyzed the data, prepared the table and all figures and wrote the main manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eI am grateful to Professor Christopher J. Elson of the University of Bristol for scientific advice and assistance and Professor Neil Williams of the University of Bristol for donating the recombinant EtxB.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbas, A. K., Murphy, K. M., \u0026amp; Sher, A. (1996). Functional diversity of helper T lymphocytes. \u003cem\u003eNature\u003c/em\u003e, \u003cem\u003e383\u003c/em\u003e(6603), 787\u0026ndash;793. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/383787A0\u003c/span\u003e\u003cspan address=\"10.1038/383787A0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAman, A. T., Fraser, S., Merritt, E. A., Rodigherio, C., Kenny, M., Ahn, M., Hol, W. G. J., Williams, N. A., Lencer, W. I., \u0026amp; Hirst, T. R. (2001). 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Subsets of regulatory T cells and their roles in allergy. \u003cem\u003eJournal of Translational Medicine\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(1), 125. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1479-5876-12-125\u003c/span\u003e\u003cspan address=\"10.1186/1479-5876-12-125\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-umm-alqura-university-for-medical-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Journal of Umm Al-Qura University for Medical Science](https://link.springer.com/journal/44361)","snPcode":"44361","submissionUrl":"https://submission.springernature.com/new-submission/44361/3","title":"Journal of Umm Al-Qura University for Medical Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"EtxB, Autoimmune hemolytic anemia, NZB mice","lastPublishedDoi":"10.21203/rs.3.rs-7656673/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7656673/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe B subunit of \u003cem\u003eEscherichia coli\u003c/em\u003e heat-labile enterotoxin (EtxB) is a strong immunological modulator that enhances T helper-1 (Th1)-mediated autoimmune diseases. This study aimed to evaluate the effectiveness of inhaled EtxB in treating autoimmune hemolytic anemia (AIHA) in New Zealand Black (NZB) mice, which naturally develop AIHA.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEight NZB mice received 20 \u0026micro;g of recombinant EtxB intranasally for four consecutive days, while the control group inhaled phosphate-buffered saline (PBS). The levels of total IgG, IgG1, and IgG2a antibodies bound to red blood cells (RBC) were measured by cellular ELISA at 6 and 12 weeks. Hematocrit values were assessed at 12 weeks and 4 months using a Hawksley Micro-hematocrit reader.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter 6 weeks, EtxB-treated mice exhibited higher levels of RBC-bound IgG and IgG2a, along with a significantly lower IgG1/IgG2a ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to PBS-treated controls. However, this difference was not maintained at 12 weeks (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating a temporary immune modulation. EtxB did not delay the onset of anemia, as hematocrit readings showed no significant differences between groups at any time point (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study demonstrated that inhaling EtxB temporarily modulated the immune response at 6 weeks but did not sustain this effect beyond 12 weeks or prevent anemia.\u003c/p\u003e","manuscriptTitle":"Escherichia coli enterotoxin modulates immunity but fails to prevent autoimmune hemolytic anemia in NZB mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-20 11:21:15","doi":"10.21203/rs.3.rs-7656673/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-30T19:02:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-29T22:36:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T20:18:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T15:20:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-22T08:10:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221109198924638377538274532607986800897","date":"2025-10-15T15:03:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-14T04:46:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"263286263392349016943900607699521130970","date":"2025-10-13T12:09:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322643358421595616164900236383135071147","date":"2025-10-13T12:00:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174840928811313646875038712507652509599","date":"2025-10-12T16:04:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-10T17:08:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4488692550146717192009774126716256028","date":"2025-10-10T12:42:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170154369050932776834105095823655581790","date":"2025-10-09T01:43:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235416817132457089233399167003657890937","date":"2025-10-08T16:09:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322554549237927464814013250123855595154","date":"2025-10-08T07:15:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-07T18:30:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-07T07:37:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Umm Al-Qura University for Medical Science","date":"2025-10-05T17:44:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-umm-alqura-university-for-medical-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Journal of Umm Al-Qura University for Medical Science](https://link.springer.com/journal/44361)","snPcode":"44361","submissionUrl":"https://submission.springernature.com/new-submission/44361/3","title":"Journal of Umm Al-Qura University for Medical Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6a6abc84-cc8e-45c2-910f-4615c7a7702d","owner":[],"postedDate":"October 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-21T18:39:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-20 11:21:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7656673","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7656673","identity":"rs-7656673","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}