Macrophage migration inhibitory factor plays a critical role in mediating protection against the helminth parasite Taenia crassiceps

To determine the role of endogenous migration inhibitory factor (MIF) in regulation of immune response during murine cysticercosis caused by the helminth parasite Taenia crassiceps, we analyzed the course of T. crassiceps infection in MIF(-/-) BALB/c mice. MIF(-/-) mice were highly susceptible to T. crassiceps and developed significantly higher parasite loads compared to similarly infected MIF(+/+) mice. Throughout the course of infection, Taenia crassiceps soluble antigen-stimulated spleen cells from both MIF(+/+) and MIF(-/-) mice produced significant and comparable levels of interleukin-4 (IL-4), but those from MIF(-/-) mice produced significantly more IL-13, as well as gamma interferon (IFN-gamma), suggesting that the susceptibility of MIF(-/-) mice to T. crassiceps was not due to the lack of IFN-gamma production. Interestingly, low levels of both total and specific immunoglobulin G2a were observed in MIF(-/-) cysticercotic mice despite the high IFN-gamma levels; in addition, peritoneal macrophages obtained from T. crassiceps-infected MIF(-/-) mice at different time points failed to respond efficiently to stimulation in vitro with lipopolysaccharide plus IFN-gamma and produced significantly lower levels of IL-12, tumor necrosis factor alpha, and NO compared to those from MIF(+/+) mice. These findings demonstrate that MIF plays a critical role in mediating protection against T. crassiceps in vivo. Moreover, these findings also suggest that impaired macrophage function rather than the lack of Th1 development may be responsible for mediating susceptibility to T. crassiceps.     

Development of protective immunity against cutaneous leishmaniasis is dependent on STAT1-mediated IFN signaling pathway

Although STAT1-dependent signaling mediates biological functions of IFN-alpha/beta and IFN-gamma, recent reports indicate that STAT1-independent IFN signaling also regulates expression of several genes. To determine the roles of STAT1-dependent and -independent IFN signaling in the regulation of immunity during cutaneous leishmaniasis, we studied the course of Leishmania major infection in resistant C57BL/6 mice lacking the STAT1 gene. While L. major-infected STAT1(+/+) mice resolved their lesions, STAT1(-/-) mice developed large lesions containing significantly more parasites. Moreover, the inability of STAT1(-/-) mice to control L. major infection was due to the lack of Th1 development associated with reduced production of IL-12, IFN-gamma and nitric oxide. Although STAT1(-/-) mice produced more IL-4 and total IgE than STAT1(+/+) mice later during infection, these differences were not significant. Nevertheless, at these time points lymph node cells from STAT1(-/-) mice produced significantly more IL-10. Finally, STAT1(-/-) mice were also susceptible to low dose L. major infection. Thesefindings demonstrate that STAT1-mediated IFN signaling is indispensable for the development of protective immunity against cutaneous L. major infection. Moreover, they also suggest that the protective role of STAT1-mediated signaling is due to its ability to induce Th1 development during infection with this parasite.     

Migration-inhibitory factor gene-deficient mice are susceptible to cutaneous Leishmania major infection

To determine the role of endogenous migration-inhibitory factor (MIF) in the development of protective immunity against cutaneous leishmaniasis, we analyzed the course of cutaneous Leishmania major infection in MIF gene-deficient mice (MIF(-/-)) and wild-type (MIF(+/+)) mice. Following cutaneous L. major infection, MIF(-/-) mice were susceptible to disease and developed significantly larger lesions and greater parasite burdens than MIF(+/+) mice. Interestingly, antigen-stimulated lymph node cells from MIF(-/-) mice produced more interleukin-4 (IL-4) and gamma interferon (IFN-gamma) than those from MIF(+/+) mice, although the differences were statistically not significant. IFN-gamma-activated resting peritoneal macrophages from MIF(-/-) mice showed impaired macrophage leishmanicidal activity and produced significantly lower levels of nitric oxide and superoxide in vitro. The macrophages from MIF(-/-) mice, however, produced much more IL-6 than macrophages from wild-type mice. These findings demonstrate that endogenous MIF plays an important role in the development of protective immunity against L. major in vivo. Furthermore, they indicate that the susceptibility of MIF(-/-) mice to L. major infection is due to impaired macrophage leishmanicidal activity rather than dysregulation of Th1 and Th2 responses.     

Chronic helminth infection induces alternatively activated macrophages expressing high levels of CCR5 with low interleukin-12 production and Th2-biasing ability

Helminth infections induce Th2-type biased immune responses. Although the mechanisms involved in this phenomenon are not yet clearly defined, antigen-presenting cells (APC) could play an important role in this process. Here, we have used peritoneal macrophages (F4/80+) recruited at different times after challenge with Taenia crassiceps as APC and tested their ability to regulate Th1/Th2 differentiation. Macrophages from acute infections produced high levels of interleukin-12 (IL-12) and nitric oxide (NO), paralleled with low levels of IL-6 and prostaglandin E(2) (PGE(2)) and with the ability to induce strong antigen-specific CD4+ T-cell proliferation in response to nonrelated antigens. In contrast, macrophages from chronic infections produced higher levels of IL-6 and PGE(2) and had suppressed production of IL-12 and NO, associated with a poor ability to induce antigen-specific proliferation in CD4+ T cells. Failure to induce proliferation was not due to a deficient expression of accessory molecules, since major histocompatibility complex class II, CD40, and B7-2 were up-regulated, together with CD23 and CCR5 as infection progressed. These macrophages from chronic infections were able to bias CD4+ T cells to produce IL-4 but not gamma interferon (IFN-gamma), contrary to macrophages from acute infections. Blockade of B7-2 and IL-6 and inhibition of PGE(2) failed to restore the proliferative response in CD4+ T cells. Furthermore, studies using STAT6(-/-) mice revealed that STAT6-mediated signaling was essential for the expansion of these alternatively activated macrophages. These data demonstrate that helminth infections can induce different macrophage populations that have Th2-biasing properties.     

Complement C5a anaphylatoxin is an innate determinant of dendritic cell-induced Th1 immunity to Mycobacterium bovis BCG infection in mice

During acquired immunity to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection in mice, dendritic cells (DCs) present mycobacterial antigens to naive T cells to prime an immune response. Complement C5a (anaphylatoxin) secreted by mycobacteria-infected macrophages regulates IL-12p70 production. As IL-12p70 regulates Th1 immunity against mycobacteria in mice, we examined the effects of C5a on IL-12p70 secretion by murine DCs and Th1 immunity. DCs cultured from C5-deficient (C5(-/-)) and -sufficient (C5(+/+)) mice were infected with BCG in the presence or absence of the C5a peptide. ELISA showed that C5(-/-) DCs secreted less IL-12p70 (600 pg/mL vs. 100 pg/mL) than C5(+/+) DCs, and they secreted more IL-10. Using immunophenotyping, reduced CD40 expression was found on C5(-/-) DCs after BCG infection. BCG-primed DCs were then cocultured with naive or BCG-immune T cells to differentiate them into IFN-gamma-secreting Th1 T cells. Coincident with increased IL-12p70 levels, BCG-primed C5(+/+) DCs cocultured with naive or immune C5(+/+) T cells showed a larger increase in CD4+ IFN-gamma/CD8+ IFN-gamma+ T cells compared with cocultured DCs and T cells from C5(-/-) mice. Thus, BCG-primed C5(+/+) DCs were better able to drive a Th1 response. Furthermore, BCG aerosol-infected C5(-/-) mice showed reduced CD4 and CD8 IFN-gamma-secreting T cells in the lungs, concurrent with an increased growth of BCG. Thus, C5a, an innate peptide, appears to play an important role in the generation of acquired immune responses in mice by regulating the Th1 response through modulation of IL-12p70 secretion from DCs.     

A regulatory role for suppressor of cytokine signaling-1 in T(h) polarization in vivo

Suppressor of cytokine signaling (SOCS)-1 is an inhibitory molecule for JAK, and its deficiency in mice leads to lymphocyte-dependent multi-organ disease and perinatal death. Crossing of SOCS-1(-/-) mice on an IFN-gamma(-/-), STAT1(-/-) and STAT6(-/-) background revealed that the fatal disease of SOCS-1(-/-) mice is also dependent on IFN-gamma/STAT1 and IL-4/STAT6 signaling pathways. Since IFN-gamma and IL-4 are representative T(h)1 and T(h)2 cytokines respectively, here we investigated the role of SOCS-1 in T(h) differentiation. Freshly isolated SOCS-1(-/-) CD4(+) T cells stimulated with anti-CD3 rapidly produced larger amounts of IFN-gamma and IL-4 than control cells, suggesting that these mutant T cells had already differentiated into T(h)1 and T(h)2 cells in vivo. In addition, SOCS-1(+/-) CD4(+) T cells cultured in vitro produced significantly larger amounts of IFN-gamma and IL-4 than SOCS-1(+/+) cells. Similarly, SOCS-1(+/-) CD4(+) T cells produced more IFN-gamma and IL-4 than SOCS-1(+/+) cells after infection with Listeria monocytogenes and Nippostrongyrus braziliensis respectively. Since IL-12-induced STAT4 and IL-4-induced STAT6 activation is sustained in SOCS-1(-/-) T cells, the enhanced T(h) functions in SOCS-1(-/-) and SOCS-1(+/-) mice appear to be due to the enhanced effects of these cytokines. These results suggest that SOCS-1 plays a regulatory role in both T(h)1 and T(h)2 polarizations.     

Vaccine-induced protection against Leishmania amazonensis is obtained in the absence of IL-12/23p40

Protozoa of the genus Leishmania are intracellular parasites of macrophages and may cause diverse clinical forms of leishmaniasis, including cutaneous, diffuse cutaneous, mucocutaneous and visceral leishmaniasis. Infection with L. major in mice indicates that a protective immune response is achieved when Th1 cells are developed. Thus, adoptive or vaccine-induced protection against leishmaniasis is largely dependent on cell-mediated immunity and IFN-gamma production. Induction of a Th1 response is dependent on the presence of IL-12 whilst lymphocytes are activated. This study was aimed at evaluating the role of IL-12 during infection with L. amazonensis and after vaccination with Leishvacin (killed Leishmania amazonensis promastigotes), since the role of this cytokine in vaccine-induced immunity with this preparation in experimental models or in humans is not yet elucidated. Hence, C57BL/6 interleukin-12-deficient mice (IL-12p40(-/-)) and wild-type controls (wt) were infected with L. amazonensis and the course of infection, parasite burden and cytokine production were compared. IL-12p40(-/-) mice were more susceptible to L. amazonensis than wt: lesions and parasite burden were larger in IL-12p40(-/-) when compared to wt. Interestingly, IL-4 was not produced in the absence of IL-12 in response to infection with L. amazonensis. To evaluate the role of IL-12 in the vaccine-induced immunity against L. amazonensis infection, IL-12p40(-/-) wt mice were vaccinated in the base of the tail and subsequently challenged with L. amazonensis in the footpads. Surprisingly, vaccinated IL-12p40(-/-) mice developed smaller lesions and had fewer parasites in footpads than non-vaccinated controls. Lymph node and spleen cells from vaccinated IL-12p40(-/-) mice did not produce high levels of IFN-gamma in response do in vitro stimulus with antigen. Hence, partial protection against infection with L. amazonensis could be obtained in the absence of functional IL-12 and a typical Th1 response.     

Interleukin-27R (WSX-1/T-cell cytokine receptor) gene-deficient mice display enhanced resistance to leishmania donovani infection but develop severe liver immunopathology

The interleukin-27 (IL-27)/T-cell cytokine receptor (TCCR) pathway plays an important role in development of protective immunity against cutaneous leishmaniasis caused by Leishmania major. In this study, we analyzed the role of IL-27/TCCR pathway in the host defense against visceral leishmaniasis (VL) by monitoring the course of L. donovani infection in TCCR-deficient C57BL/6 (TCCR-/-) mice. TCCR-/- mice mounted a robust inflammatory response, produced high levels of pro-inflammatory cytokines, and developed severe liver pathology after L. donovani infection that eventually resolved. Interestingly, L. donovani-infected TCCR-/- mice controlled the parasite growth in their organs significantly faster than similarly infected TCCR+/+ mice. Adoptive cell transfer and cell depletion studies revealed that CD4(+) T cells were involved in mediating liver immunopathology and controlling L. donovani growth in TCCR-/- mice. These results indicate that the IL-27/TCCR pathway is not essential for the induction of protective Th1 response during VL but is involved in mediating susceptibility to L. donovani. Additionally, the data demonstrate that although the IL-27/TCCR interaction limits the severity of liver inflammation during VL by controlling CD4(+) T-cell activity, it is not required for the resolution of hepatic immunopathology.     

STAT-4 mediated IL-12 signaling pathway is critical for the development of protective immunity in cutaneous leishmaniasis.

Recent studies have demonstrated that two IL-12 signaling pathways, a STAT 4 - dependent and STAT4 - independent, are involved in the development of a Th1-like response. To determine their roles in the development of protective immunity against Leishmania major, we monitored progression of cutaneous Leishmania major infection in STAT4-deficient mice (STAT4-/-) compared to similarly infected wild-type (STAT4+/+) mice. Although the onset of lesion growth was delayed in STAT4-/- mice during the early phase of infection, these mice eventually developed large, non-healing lesions, whereas STAT4+/+ mice resolved their lesions. As infection progressed, both STAT4+/+ and STAT4-/- mice infected with L. major displayed similar titers of Leishmania-specific IgG1 and IgE but later produced lower IgG2a. On days 20 and 40 post-infection, Leishmania antigen-stimulated lymphnode cells from STAT4-/- mice produced significantly lower amounts of IFN-gamma than those from STAT4+/+ mice as measured by enzyme-linked immunosorbent assay. There was no significant difference, however, in IL-4 and IL-12 production between the two groups. These results indicate that STAT4-mediated IL-12 signaling is critical for the development of protective Th1 response following L. major infection in genetically resistant mice. Additionally, they demonstrate that, although genetically resistant mice lacking STAT4 signaling pathway develop large, non-healing lesions, they do not default towards a Th2-like response.     

Genital tract infection with Chlamydia trachomatis fails to induce protective immunity in gamma interferon receptor-deficient mice despite a strong local immunoglobulin A response.

CD4+ T cells have been found to play a critical role in immune protection against Chlamydia trachomatis infection. Since both humoral and cell-mediated antichlamydial immunity have been implicated in host protection, the crucial effector functions provided by the CD4+ T cells may rely on Th1 or Th2 functions or both. In the present study, we evaluated the development of natural immunity following vaginal infection with C. trachomatis serovar D in female gamma interferon receptor-deficient (IFN-gammaR-/-) mice with a disrupted Th1 effector system. We found that in comparison with wild-type mice, the IFN-gammaR-/- mice exhibited a severe ascending primary infection of prolonged duration which stimulated almost 10-fold-stronger specific local immunoglobulin A (IgA) and IgG responses in the genital tract. Following resolution of the primary infection and despite the augmented antibody responses to chlamydiae, the IFN-gammaR-/- mice were completely unprotected against reinfection, suggesting that local antibodies play a subordinate role in host protection against chlamydial infection. Immunohistochemical analysis of frozen sections of the genital tract revealed many CD4+ T cells in the IFN-gammaR-/- mice, with a dominance of interleukin 4-containing cells in mice following resolution of the secondary infection. However, in contrast to the findings with wild-type mice, the typical clusters of CD4+ T cells were not found in the IFN-gammaR-/- mice. Few and similarly distributed CD8+ T cells were observed in IFN-gammaR-/- and wild-type mice. Whereas chlamydia-infected macrophages from wild-type mice had no inclusion bodies (IB) and produced significant amounts of nitric oxide (NO) in the presence of IFN-gamma, macrophages from IFN-gammaR-/- mice contained many IB but no NO. These results indicate that CD4+ Th1 cells and IFN-gamma, rather than local antibodies, are critical elements in host immune protection stimulated by a natural ascending C. trachomatis infection in the female genital tract.     

Involvement of CD4(+) Th1 cells in systemic immunity protective against primary and secondary challenges with Trypanosoma cruzi

In general, gamma interferon (IFN-gamma)-producing CD4(+) Th1 cells are important for the immunological control of intracellular pathogens. We previously demonstrated an association between parasite-specific induction of IFN-gamma responses and resistance to the intracellular protozoan Trypanosoma cruzi. To investigate a potential causal relationship between Th1 responses and T. cruzi resistance, we studied the ability of Th1 cells to protect susceptible BALB/c mice against virulent parasite challenges. We developed immunization protocols capable of inducing polarized Th1 and Th2 responses in vivo. Induction of parasite-specific Th1 responses, but not Th2 responses, protected BALB/c mice against virulent T. cruzi challenges. We generated T. cruzi-specific CD4(+) Th1 and Th2 cell lines from BALB/c mice that were activated by infected macrophages to produce their corresponding cytokine response profiles. Th1 cells, but not Th2 cells, induced nitric oxide production and inhibited intracellular parasite replication in T. cruzi-infected macrophages. Despite the ability to inhibit parasite replication in vitro, Th1 cells alone could not adoptively transfer protection against T. cruzi to SCID mice. In addition, despite the fact that the adoptive transfer of CD4(+) T lymphocytes was shown to be necessary for the development of immunity protective against primary T. cruzi infection in our SCID mouse model, protective secondary effector functions could be transferred to SCID mice from memory-immune BALB/c mice in the absence of CD4(+) T lymphocytes. These results indicate that, although CD4(+) Th1 cells can directly inhibit intracellular parasite replication, a more important role for these cells in T. cruzi systemic immunity may be to provide helper activity for the development of other effector functions protective in vivo.     

Resistance to acute babesiosis is associated with interleukin-12- and gamma interferon-mediated responses and requires macrophages and natural killer cells

We examined the role of the cytokines gamma interferon (IFN-gamma) and interleukin-12 (IL-12) in the model of acute babesiosis with the WA1 Babesia. Mice genetically deficient in IFN-gamma-mediated responses (IFNGR2KO mice) and IL-12-mediated responses (Stat4KO mice) were infected with the WA1 Babesia, and observations were made on the course of infection and cytokine responses. Levels of IFN-gamma and IL-12 in serum increased 24 h after parasite inoculation. The augmented susceptibility observed in IFNGR2KO and Stat-4KO mice suggests that the early IL-12- and IFN-gamma-mediated responses are involved in protection against acute babesiosis. Resistance appears to correlate with an increase in nitric oxide (NO) production. In order to assess the contribution of different cell subsets to resistance against the parasite, we also studied mice lacking B cells, CD4+ T cells, NK cells, and macrophages. Mice genetically deficient in B lymphocytes or CD4+ T lymphocytes were able to mount protective responses comparable to those of immunosufficient mice. In contrast, in vivo depletion of macrophages or NK cells resulted in elevated susceptibility to the infection. Our observations suggest that a crucial part of the response that protects from the pathogenic Babesia WA1 is mediated by macrophages and NK cells, probably through early production of IL-12 and IFN-gamma, and induction of macrophage-derived effector molecules like NO.     

Immune responses associated with susceptibility of C57BL/10 mice to Leishmania amazonensis.

Leishmaniae are protozoans which, depending upon both the host and parasite species, can cause either a healing or nonhealing infection. While C57BL/10 mice are able to heal following infection with Leishmania major, they fail to heal following infection with Leishmania amazonensis. In order to address the role of Th1 and Th2 cell responses in the outcome of these infections in C57BL/10 mice, gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production was assessed. While cells from L. major-infected C57BL/10 mice produced high levels of IFN-gamma, cells from L. amazonensis-infected animals produced little or no IFN-gamma. On the other hand, IL-4 was produced only by cells from L. amazonensis-infected C57BL/10 mice, but this production was restricted to the first few weeks of infection. Later in infection, when lesions were evident, no IL-4 was detected. Treatment of BALB/c mice with a monoclonal antibody (11B11) directed against IL-4 induced a dramatic reduction in L. amazonensis lesions. This reduction was associated with a decrease in IL-4 levels and an increase in IFN-gamma production. However, only a slight reduction in lesion sizes and parasite numbers was observed when anti-IL-4-treated C57BL/10 mice were infected with L. amazonensis. These results suggest that IL-4 may have an important role in mediating susceptibility to L. amazonensis in BALB/c mice, as previously demonstrated for L. major. More importantly, however, the data suggest that susceptibility to L. amazonensis in C57BL/10 mice is due to the absence of a Th1 cell response, rather than to the presence of a Th2 cell response.     

Interleukin-4 and interleukin-10 are involved in host resistance to Staphylococcus aureus infection through regulation of gamma interferon

Our previous study showed that gamma interferon (IFN-gamma), a T-helper 1 (Th1)-type cytokine, plays a detrimental role in Staphylococcus aureus infection in mice. In this study, the role of Th2-type cytokines such as interleukin-4 (IL-4) and IL-10 in S. aureus infection was investigated. IL-10 mRNA was induced in parallel with IFN-gamma in the spleens and kidneys of mice during S. aureus infection, whereas IL-4 mRNA was induced in the spleens but not in the kidneys of these animals. Spleen cells obtained from S. aureus-infected mice produced lower titers of IFN-gamma and higher titers of IL-4 and IL-10 in response to heat-killed S. aureus than did those from uninfected mice. Administration of anti-IL-4 monoclonal antibody (MAb) or anti-IL-10 MAb inhibited the elimination of S. aureus cells from the kidneys of mice. IFN-gamma mRNA expression was enhanced in the spleens of anti-IL-4 MAb- or anti-IL-10 MAb-treated mice and also in the kidneys of anti-IL-4 MAb-treated animals. Next, we evaluated the role of IFN-gamma in S. aureus infection in IFN-gamma(-/-) mice. An increase in survival rates, a decrease in bacterial numbers in the kidneys, and an amelioration of histologic abnormalities in these organs were observed in IFN-gamma(-/-) mice compared with those in IFN-gamma(+/+) mice. Administration of MAb against IL-4 or IL-10 failed to affect bacterial growth in the spleens and kidneys of IFN-gamma(-/-) mice irrespective of the expression of Th2 response. These results suggest that S. aureus infection induced a Th2 response and that IL-4 and IL-10 might play a protective role through the regulation of IFN-gamma in S. aureus infection.     

Immune effector mechanism in parasitic infections

In this article is a summary of our recent findings on the role of nitric oxide (NO) as an effector mechanism against the intracellular parasite, Leishmania major. NO is produced in large amounts in murine macrophages following activation by IFNgamma synthesized by Th1 cells. NO production is inhibited by IL-4, a product of Th2 cells. A set of stable cell surface markers has now been identified. ST2L and IL-18R are selectively expressed on Th2 and Th1 cells respectively. Antibody against ST2L can down-regulate Th2 cells in the highly susceptible BALB/c mice leading to control of otherwise fatal L. major infection. These results show directly the critical role of the balance between Th1 and Th2 cells in cutaneous leishmaniasis.     

Cruzipain induces both mucosal and systemic protection against Trypanosoma cruzi in mice

Cruzipain, the major cysteinyl proteinase of Trypanosoma cruzi, is expressed by all developmental forms and strains of the parasite and stimulates potent humoral and cellular immune responses during infection in both humans and mice. This information suggested that cruzipain could be used to develop an effective T. cruzi vaccine. To study whether cruzipain-specific T cells could inhibit T. cruzi intracellular replication, we generated cruzipain-reactive CD4(+) Th1 cell lines. These T cells produced large amounts of gamma interferon when cocultured with infected macrophages, resulting in NO production and decreased intracellular parasite replication. To study the protective effects in vivo of cruzipain-specific Th1 responses against systemic T. cruzi challenges, we immunized mice with recombinant cruzipain plus interleukin 12 (IL-12) and a neutralizing anti-IL-4 MAb. These immunized mice developed potent cruzipain-specific memory Th1 cell responses and were significantly protected against normally lethal systemic T. cruzi challenges. Although cruzipain-specific Th1 responses were associated with T. cruzi protective immunity in vitro and in vivo, adoptive transfer of cruzipain-specific Th1 cells alone did not protect BALB/c histocompatible mice, indicating that additional immune mechanisms are important for cruzipain-specific immunity. To study whether cruzipain could induce mucosal immune responses relevant for vaccine development, we prepared recombinant attenuated Salmonella enterica serovar Typhimurium vaccines expressing cruzipain. BALB/c mice immunized with salmonella expressing cruzipain were significantly protected against T. cruzi mucosal infection. Overall, these data indicate that cruzipain is an important T. cruzi vaccine candidate and that protective T. cruzi vaccines will need to induce more than CD4(+) Th1 cells alone.     

Immunoglobulin A-deficient mice exhibit altered T helper 1-type immune responses but retain mucosal immunity to influenza virus

We have previously demonstrated that immunoglobulin A (IgA)(-/-) knockout (KO) mice exhibit levels of susceptibility to influenza virus infection that are similar to those of their normal IgA(+/+) littermates. To understand the mechanism of this apparent mucosal immunity without IgA, immunoglobulin isotype and T helper 1 (Th1)-type [interferon-gamma (IFN-gamma)] and Th2-type [interleukin (IL)-4, IL-5)] cytokine responses to influenza vaccine were evaluated. Intranasal immunization with influenza virus subunit vaccine plus cholera toxin/cholera toxin B subunit (CT/CTB) induced significant influenza virus-specific immunoglobulin G (IgG) antibody in the serum and nasal passages of both IgA(-/-) and IgA(+/+) mice, while IgA antibodies were induced only in IgA(+/+) mice. IgA KO mice exhibited an IgG1 subclass haemagglutinin (HA)-specific response but no detectable IgG2a and IgG2b responses. In contrast, IgA(+/+) mice exhibited significant IgG1 as well as IgG2a responses. This indicates a predominant Th2-type response in IgA KO mice compared to normal mice. Following stimulation with influenza virus in vitro, splenic lymphocytes from immunized IgA(-/-) mice produced significantly lower levels of IFN-gamma than IgA(+/+) mice (P < 0.001), but elaborated similar levels of IL-4 and IL-5. This was true at both protein and mRNA levels. Immunized mice were challenged intranasally with a small inoculum of influenza virus to allow deposition of virus in the nasal mucosal passages. Compared to non-immunized mice, immunized IgA(-/-) and IgA(+/+) mice exhibited significant, but similar levels of reduction in virus titres in the nose and lung. These results demonstrate that in addition to IgA deficiency, IgA gene deletion also resulted in down-regulated Th1-type immune responses and confirm our previous data that IgA antibody is not indispensable for the prevention of influenza virus infection.